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Technical Advances |
,
,¶,
From the Departments of Oncology *
and
Pathology,
the Cambridge Institute for
Medical Research/Wellcome Trust Centre for Molecular Mechanisms in
Disease,
and Strangeways Research
Laboratories,¶
University of Cambridge, Cambridge;
and the Cambridge Breast Unit,
Addenbrooke’s
Hospital, Cambridge, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionConclusionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionConclusionReferences |
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionConclusionReferences |
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Eighteen human breast cancer cell lines, KPL-1, VP185, VP229, VP267, VP303, SUM149, SUM159, SUM185, SUM225, OCUB-F, OCUB-M, Matu, MT-1, MT-3, CAL51, MDA-MB157, MDA-MB175, and MCF7, were grown in monolayers in medium supplemented with 5 to 10% fetal bovine serum. Extraction of DNA from the cancer cell lines was performed using DNAzol Reagent (Life Technologies Ltd., Paisley, UK).
Primary breast tumor samples were obtained from 10 patients. The use of
clinical materials for research was approved by the Research Ethics
Committee. Cancer cells were obtained by laser capture microdissection
(PixCell II laser capture microdissection system; Arcturus Engineering,
Inc., Mountain View, CA).7
Captured cells (
500) were
immediately resuspended in 20 µl of buffer containing 10 mmol/L
Tris-HCl, pH 8.3, 2.5 mmol/L MgCl2, 50 mmol/L KCl, 0.45%
Nonidet P-40, 0.45% Tween 20, 0.1 mg/ml proteinase K, and were
incubated overnight at 55°C. The mixture was boiled for 10 minutes to
inactivate the proteinase K and was used for DOP-PCR.
DOP-PCR
DOP-PCR was performed according to our published protocol8 with modifications for formalin-fixed tissue. PCR was performed on a thermocycler (model PTC-225; MJ Research, Inc., Watertown, MA) in two separate phases. In the preamplification step, the eight initial cycles were performed at low stringency conditions (denaturation at 94°C for 90 seconds, annealing at 30°C for 180 seconds, ramp from 30 to 72°C for 210 seconds, and extension at 72°C for 180 seconds), followed by 25 cycles in high stringency conditions (denaturation at 94°C for 90 seconds, annealing at 62°C for 90 seconds, and extension at 72°C for 90 seconds; a final extension for 480 seconds at 72°C followed). This primary DOP-PCR product is labeled in a second amplification step for 28 cycles in high stringency conditions mentioned above. UN1 primer (5'-CCG ACT CGA GNN NNN NAT GTG G-3', with N = A, C, G, T) was used in both reactions.
Array-CGH
Array-CGH was performed following the original protocol for nick-translation3 and using a protocol modified to use DOP-PCR product. Briefly, normal female DNA (normal reference DNA) was labeled by nick translation with Alexa Fluor 594-5-dUTP (Molecular Probes, Leiden, The Netherlands) and the tumor DNAs with Alexa Fluor 488-5-dUTP (Molecular Probes). Direct red/green-fluorescence labeling (594-5-dUTP or 488-5-dUTP) of the same DNA samples was also performed by two-step DOP-PCR as mentioned above. Labeled test DNA (0.5 µg of nick-translated DNA or 1.5 µg of the labeled DOP-PCR product) and normal reference DNA (0.5 µg or 1.5 µg, respectively), were mixed in hybridization buffer (Vysis, Inc., Downers Grove, IL), and hybridized to AmpliOnc I DNA array (Vysis, Inc.). This chip contains 59 clones from 57 oncogenes (BCL2 and AR are represented by both 5' and 3' genomic clones) representing genomic regions that have been reported to be amplified in human tumors. We made two hybridization combinations (except where indicated otherwise): nick-labeled test DNA versus nick-labeled reference DNA (standard array CGH hybridization) and DOP-PCR-labeled test DNA versus DOP-PCR-labeled reference DNA (DOP-PCR-based array CGH). Arrays were hybridized for 24 to 48 hours, washed, and counterstained with the 4,6-diamidino-2-penylindole (DAPI) IV mounting solution.
Imaging and Analysis
Arrays were analyzed using GenoSensor Reader System (Vysis, Inc.). The filter set used in this system is: blue light excitation filter, 405/11 nm; green light, 490/17 nm; red light, 570/20 nm; and triple bandpass emission filter, 463/25 nm, 530/30 nm, and 615/30 nm. Signals from reference and test DNA (594-5-dUTP and 488-5-dUTP, respectively) were quantitatively detected with exposure times of 0.5 to 20 seconds by autoexposure system. Images were analyzed with custom software that segmented the array targets based on the DAPI staining, estimated and subtracted the background in the green and red fluorescence images, and calculated the total intensity and the intensity ratio of green and red signals for each target (three replicate spots for each target gene). The Pearson’s r correlation of a scatterplot of the test versus reference signal intensities for the pixels in each target was calculated. Data from targets with r values below 0.8 were discarded. The test/reference ratios are defined as ratio of the sum of test intensity pixel values to the sum of reference intensity pixel values. For each target ratios on replicate targets with r > 0.8 were averaged.
Control Amplicon DNA
To test the performance of the protocols and system used for amplification detection we used a mixture of DNAs (CoSH) extracted from three tumor cell lines (Vysis Inc.). The CoSH DNA is mixed as follows: COLO 320 (colon cancer), 35%; SJSA-1 (sarcoma), 40%; and BT-474 (breast cancer), 25%.
FISH
Metaphase chromosomes were prepared by standard methods. The nine breast cancer cell lines used for this study were: KPL-1, VP229, SUM159, OCUB-F, Matu, MT-1, MT3, CAL51, and MDA-MB175. Slides were denatured for 90 seconds in 70% formamide/2x standard saline citrate at 72°C and immediately snap-cooled in ice-cold ethanol series. Denatured centromeric and single-locus FISH probes in dual color combinations were hybridized according to a standard protocol; and where relevant, detected with fluorescein isothiocyanate-conjugated anti-digoxigenin antibody. The following genes were studied: ERBB2 (Appligene Oncor, Tucson, AZ), ZNF217 (Vysis Inc.), MYC (Appligene Oncor or Vysis Inc.), and CCND1 (Vysis, Inc.), as well as a centromeric probe for chromosome 17 as a control. FISH experiments using touch preparation of frozen breast cancer tissues were also performed. The copy number index for each oncogene was calculated as follows: (average gene copy number)/(modal chromosome number) x 23. The average gene copy number of 50 to 100 interphase nuclei and/or metaphases by FISH, and the modal chromosome number calculated by 24-color FISH karyotyping, interphase FISH, and from previously published data.9
CGH
CGH was performed as described elsewhere10 in 24 breast cancer samples (18 cancer cell lines and 6 breast cancer tissues). At least 15 metaphases were captured on a Zeiss Axioplan II fluorescent microscope using SmartCapture VP software (Digital Scientific Ltd., Cambridge, UK) and analyzed by Quips CGH analysis software (Vysis Inc.). The threshold set for gains corresponded to a mean hybridization ratio between tumor and normal of >1.2:1, and for losses of <0.8:1.
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionConclusionReferences |
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We first tested the feasibility of DOP-PCR-based array CGH by
analyzing genomic DNAs from normal lymphocytes or tumor cell lines, and
by comparing the data with that of a nick translation-based array CGH.
First we hybridized normal female DNA against the same DNA, which was
labeled with Alexa Fluor 488-5-dUTP (green fluorescence) or Alexa Fluor
594-5-dUTP (red fluorescence), respectively. We made four hybridization
combinations: 1) nick-labeled normal DNA (green) versus
nick-labeled normal DNA (red), 2) DOP-PCR-labeled normal DNA (green)
versus DOP-PCR-labeled normal DNA (red), 3) nick-labeled
normal DNA (green) versus DOP-PCR-labeled normal DNA (red),
and 4) DOP-PCR-labeled normal DNA (green) versus
nick-labeled normal DNA (red). After hybridization and washing, we
detected the average green/red fluorescence ratio on three independent
spotted genomic clones for each gene on the AmpliOnc I microarray.
These showed uniform and even hybridization for all 57 target genes on
the genomic microarray: 1) average fluorescence ratio 1.01 (SD, 0.09);
2) 1.01 (0.08); 3) 0.99 (0.07); and 4) 1.03 (0.05). These results
confirm that DOP-PCR labeling of DNA is comparable to nick translation
in diploid cells without amplifications or deletions. Next, we tested
the performance of the system and validity of our protocols in the
detection of genetic amplification using human CoSH control DNA (Vysis,
Inc.). We independently labeled genomic DNA from CoSH mixture in
green/red, and from normal female leukocytes in red/green by nick
translation or DOP-PCR. The following hybridization combinations were
done, 1) nick-labeled CoSH DNA (green) versus nick-labeled
reference DNA (red); 2) DOP-PCR-labeled CoSH DNA (green)
versus DOP-PCR-labeled reference DNA (red); 3) nick-labeled
CoSH DNA (red) versus nick-labeled reference DNA (green);
and 4) DOP-PCR-labeled CoSH DNA (red) versus DOP-PCR-labeled
reference DNA (green). The fluorescence ratios determined using
nick-translation showed almost complete concordance with the values for
all eight genes with known amplification (for example: MYC
9.9 versus 10.3, SAS/CDK4 3.3 versus
3.1, and ZNF217 2.3 versus 2.1) (Figure 1)
. The fluorescence ratio of the
amplifications detected by DOP-PCR-based array CGH, showed good
concordance with, but in some cases underestimated the ratio determined
by nick-translation labeling (for example: MYC 6.5
versus 10.3, SAS/CDK4 2.5 versus 3.1,
and ZNF217 1.4 versus 2.1). For all 57 genes on
the chip, there was a statistically significant correlation between the
fluorescence ratios obtained using the two labeling methods (Spearmans
= 0.84, P < 0.001). Moreover, comparison
between normal and inverse hybridization confirmed the presence of copy
number aberrations at all eight loci examined using both labeling
methods. There was also a significant correlation between the
fluorescence ratios from inverse hybridization experiment using the two
labeling methods (Spearmans = 0.73, P <
0.001). Having established the methodology and hardware performance, we
labeled genomic DNA from a human breast cancer cell line, KPL-1, in
green, and genomic DNA from normal female leukocytes in red by
nick-translation and DOP-PCR, independently, and co-hybridized the
labeled DNAs (green and red) to the genomic microarray (Figure 2, A
and B). The increase in
fluorescence ratio detected by DOP-PCR-based array CGH closely
approximated the ratio determined by nick-translation labeling
(examples: NRAS 5.31 versus 5.55), although in
some cases DOP-PCR-based array CGH underestimated copy number,
particularly for high-level amplifications (examples:
RPS6KB1 2.81 versus. 9.39, AIB1, 4.44
versus 8.03) (Figure 2C)
. Nevertheless, DOP-PCR-based array
CGH detected all amplifications that were found using nick-translation.
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. An
excellent correlation was observed between array CGH, using the two
different labeling methods and FISH analysis (Figure 3, C and D)
.
Analysis of the scatterplot indicated a fluorescence ratio threshold
for amplification of 1.3. Using nick-translation-based array CGH, this
threshold was 100% sensitive and specific in detecting copy number
gains (copy number index of 1.5) (Figure 3C)
, and was compatible with
the distribution of fluorescence ratios in the normal-to-normal DNA
hybridization. The threshold experimentally determined by our study
underestimates the true copy number index (1.3 versus 1.5),
however, it shows good concordance with the data from other reports of
array CGH, including different methods: matrix-based CGH and cDNA
microarray based CGH.2,4
For example, most low copy number
changes were detected as a fluorescence intensity ratio <1.5
(1.1 1.4) using matrix CGH, and average fluorescence ratio
for X-chromosomal genes in a case of 47, XXX was 1.31 (1.15 to 1.5)
using cDNA microarray-based CGH. Figure 3D
also shows that
DOP-PCR-based array CGH is highly sensitive and specific. However, the
most appropriate fluorescence ratio threshold for amplification could
not be precisely determined, with values between 1.1 and 1.2 resulting
in sensitivity and specificity of 100%.
|
. Analysis of the scatter plot
revealed that the best fluorescence ratio threshold for DOP-PCR-based
array CGH was 1.2 that had 98.4% positive predictive value, 97.6%
negative predictive value, 90.6% sensitivity, and 99.6% specificity
against the result of nick-translation. Treating the fluorescence ratio
as a continuous variable there was a statistically significant
correlation between the scores of the two methods (Spearmans =
0.68, P < 0.001). Comparing this threshold with the
distribution in fluorescence ratio of the normal-to-normal DNA
hybridization using DOP-PCR labeling confirms its suitability. We also
confirmed the suitability of these array CGH fluorescence ratio
thresholds by detecting the trisomy of chromosome 7 found by 24-color
FISH karyotyping9
in the breast cancer cell line MT-3. The
average fluorescence ratio (using three genes on chromosome 7) for
nick-translation was 1.4 (range, 1.35 to 1.46) and for DOP-PCR-based
array CGH 1.3 (range, 1.22 to 1.45). Highly amplified genes that cannot be quantitatively scored by FISH (because of diffuse distribution of signal or too many to count signal dots) were initially excluded from the comparative analysis between FISH and array CGH; however all of these were detected as amplification using array CGH. The fluorescence ratio of these high-grade amplifications detected by DOP-PCR-based array CGH, showed good concordance with, but in some cases underestimated the ratio determined by nick-translation labeling (for example: ERBB2 2.5 versus 5.5 in OCUB-F and MYC 5.05 versus 15.66 in SUM159).
The number of gene deletions detected was small, so we were unable to determine the fluorescence ratio thresholds for deletion. However, in addition to the data mentioned above, similar analysis demonstrated the ability of DOP-PCR-based array CGH to detect single-copy deletion of the X chromosome gene (Androgen Receptor, Xq11-q12) in a comparison of normal male (XY) and female (XX) genomic DNA (fluorescence ratio of 0.69). This deletion ratio is comparable to previous array CGH reports.1-4 Since most reports so far used a limited number of genetic loci (a few X chromosome genes in Turner’s syndrome samples, for example) to investigate deletion, further genetic target spots that are deleted in cancer will need to be analyzed to optimize the detection level for deletion by array CGH.
To determine whether results from the optimized DOP-PCR-based array CGH
can be reproduced on paraffin tissue samples dissected by laser capture
microdissection, we performed two experiments. First, we performed
array CGH using DOP-PCR labeling in two paraffin-embedded tissues and
using nick-translation in frozen sections from the same cases. All
frozen and paraffin tissues were dissected by laser capture
microdissection (Figure 4; A, B, and C
).
The results of DOP-PCR-based array CGH in these two
paraffin-embedded cancer samples showed significant concordance with
that of nick-translation-based array CGH in tumor frozen sections
(Spearmans = 0.74, P < 0.01) (Figure 4D)
.
Second, using touch preparations of these two frozen sections, we
analyzed the copy number of four genes (CCND1,
MYC, ERBB2, and ZNF217). Complete
concordance (100% sensitivity/specificity) between array CGH with
DOP-PCR or nick-translation labeling and FISH analysis was observed for
these four genes when we used the thresholds determined in cell lines
(data not shown). These results confirmed the ability of DOP-PCR-based
array CGH to detect amplification even in laser microdissected
paraffin-embedded samples. Recently, it was reported that the high
sensitivity of real-time PCR (with the 5'-exonuclease-based assay)
enabled the reliable and objective detection of low-level
amplifications of a single gene in as few as 50 breast cancer cells
from laser-microdissected paraffin tissue sections.11
Thus, independently this data confirms the ability to detect single
gene amplifications in DNA samples isolated from paraffin-embedded
cancer tissues.
|
Detailed Amplicon Profiling of Breast Cancer
We obtained a detailed amplicon profile using a microarray chip
containing 57 oncogenes (Table 1)
(the
detailed amplicon profile of the 57 oncogenes in Table 1
are available
on the Internet (http://www.cimr.cam.ac.uk/research/groups.htm), with
data from nick-translation and DOP-PCR-based array CGH in 18 cell lines
and two frozen breast cancer tissues, and data from DOP-PCR-based array
CGH in eight cases of paraffin-embedded cancer tissues. Twenty-four of
these samples (18 cell lines and 6 cancer tissues) were also analyzed
by metaphase CGH (Figure 5)
. In all, 379
gains covering 49 genetic loci from 21 chromosomes were observed by
array CGH (data summarized in Table 1
). Allelic gains at frequencies of
>20% were observed at 39 genetic loci. Eighteen of these genes have
been extensively studied in breast cancer (Table 1A
, column a) and we
show a similar or higher frequency of gain compared to previous
reports.12-14
This increase in frequency may be because
of the high sensitivity of our method to detect small copy number
increases, or differences in pathological stage and grade of samples.
Thirteen genes (Table 1B
, column a) constitute a group that has been
implicated in breast carcinogenesis by expression or functional
analysis, and we show that these are amplified in a proportion of
breast cancers. We detected an increase copy number of KRAS2
in 10 of 27 cases (37%) of breast cancer by both labeling methods
(fluorescence ratio ranging from 1.2 5.11) (Table 1B
, column
a). Eight of these cases did not have an accompanying increase in copy
number of adjacent genes (CCND2, 12p13), indicating a
lesser probability of regional amplification or aneuploidy for this
chromosome. A diploid cell line, CAL51 also had an increased
fluorescence ratio of KRAS2 (1.77). Such a copy number
change in KRAS2 may represent gene-specific or small region
amplification. In human breast cancers, activating mutation of the
KRAS2 gene (12p12.1) is uncommon.15
To our
knowledge, just one case of amplification of the KRAS2 gene
not associated with point mutation has previously been reported (in a
lung metastasis of rectal carcinoma).16
Our results
suggest that KRAS2 activation by amplification might be
involved in the progression of human breast cancers. Eight loci not
previously shown to be amplified in breast cancer were also identified
(Table 1C
, column a).
|
|
10%.17
In this study we detected eight cases (30%)
of regional amplification at 11q13. The array used includes
CCND1, FGF4/FGF3, and EMS1, and three
breast cancers revealed the same level of amplification for all three
genes. In the other five cases only one gene in this region was
significantly amplified. At 20q12-q13, another common amplicon, we
analyzed six genes (AIB1, STK15,
CSE1L, MYBL2, PTPN1, and
ZNF217), and found 14 cancer specimens with regional
amplification. Among these, significant gain of AIB1 was
observed in two samples, CSE1L in two samples and
ZNF217 in two samples. In recent reports18,19
using array CGH two regions of amplification within a 2-Mb region of
recurrent aberration at 20q13.2 in breast cancer were shown. The
ZNF217 gene mapped to one peak, and CYP24
(encoding vitamin D 24 hydroxylase), whose overexpression is
likely to lead to abrogation of growth control mediated by vitamin D,
mapped to the other. The presence of differentially amplified genes
raises the possibility that cells of certain tumors are susceptible to
independent amplification events in these regional amplified sequences.
Further analysis with locus-specific arrays in many cancer samples will
bring more precise information about these regional amplifications. Comparison of Array-CGH and Metaphase-CGH
CGH was developed for genome-wide analysis of DNA sequence copy number in a single experiment and has been broadly applied to human cancers. The use of metaphase chromosomes, however, limits detection of events involving small regions (<20 Mb) of the genome, resolution of closely spaced aberrations, and linking ratio changes to genomic/genetic markers. Several reports indicate that there is good correlation of data obtained by CGH (relative copy number of DNA sequence), with FISH (the number of copies of a specific DNA sequence in single nuclei), or flow cytometry (DNA content/index).20,21 However these analyses were based on a limited number of data points (sample size and/or number of probes).
We compared in 24 breast cancer samples amplifications of 57 oncogenes
detected by array CGH with data on chromosomal band amplification
obtained by CGH Figure 5
; the detailed amplicon profile of the 57
oncogenes and CGH data in Figure 4
are available on the internet.
(http://www.cimr.cam.ac.uk/research/groups.htm). In most cases with
amplicons detected by CGH there was a significant number with
amplification of oncogenes known to be within the amplicon and
represented in the microarray used (Table 1
, column b). It is not
surprising the concordance is less than perfect because the true
targets of several of the amplicons are not represented in the
microarray used. Screening by metaphase CGH has proven to be highly
useful for the first step of identification of candidate cancer-related
genes, yet more sensitive techniques, such as locus or
chromosome-specific arrays covering amplified regions may facilitate
subsequent oncogene identification.19
One would also
predict that a proportion of oncogene amplifications detected by array
CGH are not picked up by classical CGH. That is indeed the case (Table 1
, column c), with at least two genes identified to be amplified in a
significant proportion of cases (PDGFRA/4q12 and
MYB/6q22 in 21 and 30% of cases, respectively) and no
amplicon of the corresponding chromosomal bands detected by classical
CGH. Although further comparative analyses using more gene-specific
loci are required, the techniques are complementary for some loci, such
as 8q24 (MYC), 11q13, and 20q12q13.
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Conclusion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionConclusionReferences |
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Acknowledgements |
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Footnotes |
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Supported by The Cancer Research Campaign; the Uehara Memorial Foundation postdoctoral research fellowship (to Y. D.); and a Sackler Studentship, Overseas Research Studentship Award, and Cambridge Commonwealth Trust Prince of Wales Scholarship (to K. L. G.).
Y. D. and S.-F. C. contributed equally to this work.
Accepted for publication January 17, 2001.
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