Cancer Epidemiology Biomarkers & Prevention Vol. 9, 175-179, February 2000
© 2000 American Association for Cancer Research
Comparative Polymerase Chain Reaction Analysis of c-myc Amplificationon Archival Breast Fine-Needle Aspiration Materials1
JianYu Rao2,
Sophia K. Apple3,
YuShen Jin,
Suhua Lin,
Roberta K. Nieberg and
Sharon L. Hirtschowitz
Department of Pathology and Laboratory Medicine, University of California at Los Angeles Medical Center, Los Angeles, California 90095
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Abstract
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The oncogene c-myc is a key regulator of cell cycle
progression (from G1 to S phase). The amplification of
c-myc can either induce cell proliferation or apoptosis.
As a part of our ongoing effort to develop methods for multiple tumor
marker analysis, this study was carried out to determine whether
biomarkers such as c-myc amplification could be analyzed
on genetic materials collected from archival fine-needle aspiration
(FNA) smears. A novel comparative PCR analysis was used to analyze
c-myc amplification semiquantitatively. Genomic DNA was
prepared using cells obtained from archival FNA materials that had
undergone quantitative fluorescence image analysis (QFIA) for other
biomarkers. Of the 72 cases selected from 1995 for this study, 53 had
an adequate amount of DNA for analysis. A novel comparative PCR
analysis was used to analyze c-myc amplification
quantitatively. For each batch of experiments, DNA from the high
c-myc expressing cells, HL-60, and DNA from the low
expressing cells, K562, were served as positive and negative controls,
respectively. c-myc amplification was observed in 16
(94.1%) of 17 malignant lesions, 5 (41.7%) of 12 proliferative breast
diseases with nuclear atypia, and 4 (16.7%) of 24 other benign lesions
(fibroadenoma or fibrocystic disease). The overall difference of
c-myc expression among these groups was highly
significant by 
2 analysis (P =
0.0002). We conclude that multiple phenotypic markers and genotypic
markers may be combined in a risk assessment biomarker profile on small
FNA samples that can be obtained on multiple occasions relatively
noninvasively from the patient. The results of this study suggest that
c-myc amplification may be a biomarker of breast cancer
risk. However, additional large, prospective studies are needed to
confirm the current observation.
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Introduction
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FNA4
is a simple, fast, and cost-effective method for evaluating breast
lesions (1)
. However, morphological analysis alone on FNA
materials may not be accurate for defining premalignant lesions.
Detecting these lesions is important for defining an individual’s risk
for developing breast cancer (2)
. FNA materials
potentially provide several advantages over tissue samples for the
evaluation of tumor markers. These advantages include the minimally
invasive nature of the procedure and the fact that quantitative
analysis of biomarker expression can be carried out because the cells
obtained by FNA are fresh and enhancing controlled fixation and
biomarker quantitation.
The overall aim of this project was to develop a schema whereby
multiple biomarkers can be analyzed using the minimum amount of
cytological materials. Multiple biomarker analysis is essential for
individual risk assessment of breast cancer development because breast
cancer, like any other epithelial malignancy, develops through multiple
pathways. Two separate studies were carried out on the same FNA smears.
The first one was to measure multiple biomarkers (DNA ploidy,
G-actin, and p53) simultaneously on single-cell basis using
the state-of-the-art QFIA technique (3)
. The purpose of
the present study was to determine whether genomic DNA can be extracted
from the cells on the FNA smears so that biomarkers such as
c-myc amplification can be analyzed using a novel,
semiquantitative, cPCR technique. The preliminary results of the
present study will also determine whether c-myc
amplification can be used as an intermediate end-point marker for
breast cancer.
The oncogene c-myc is a key regulator of cell cycle
progression (from G1 into S phase), and its
overexpression can induce either cell proliferation or apoptosis
(4)
. Using various techniques previously, c-myc
overexpression has been detected in 20–60% of primary breast cancers
and in 60% of benign biopsies from patients who subsequently developed
breast CA (5
, 6)
. These findings indicate that
c-myc amplification may be an important biomarker for breast
cancer. However, c-myc amplification has not been evaluated
on breast FNA samples, particularly archived materials.
In this study, archived FNA smears collected from 1995 were used. These
smears had previously analyzed by QFIA analysis prior to genomic DNA
extraction for PCR analysis. The results of both of the studies
demonstrate that it is feasible to study multiple biomarkers with
different methods on archival FNA smears. Our approach of combining
in situ fluorescence labeling and image analysis with
subsequent DNA analysis using PCR technique provides a powerful
approach for analyzing multiple biomarkers for individual breast cancer
risk assessment.
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Materials and Methods
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FNA Slides and Patient Population.
A detailed description of FNA materials used for this study and the
results of a two-year follow-up study of these cases have been
published elsewhere (7)
. In brief, there were 265
incidents of breast FNA at University of California-Los Angeles Medical
Center in 1995. These FNA materials were reviewed systematically, and
the cytological interpretation of the cases was confirmed by two
cytopathologists (S. K. A. and S. L. H.). Our initial design
was to carry out two sets of QFIA analysis; each set required two
alcohol-fixed slides (one served as negative control). Therefore, only
those cases who had at least four alcohol-fixed slides were carried out
by QFIA analysis for DNA ploidy, p53, and G-actin
(3)
. This yielded a total of 72 cases (20 CA, 18 PBDA, and
34 benign) from which 56 cases had an adequate number of cells on the
smear to carry out the QFIA analysis. Of 16 cases that had an
inadequate number of cells, 3 (15%) were in the CA group, 4 (22%) in
the PBDA group, and 9 (26%) in the benign group. The difference of
proportion of inadequate cases among each group did not reach
statistical significance (P > 0.05 by
2 test).
After QFIA analysis, the smears were used for the analysis of
c-myc amplification using cPCR technique. The QFIA analysis
was strictly limited on the epithelial cells. To minimize the
contamination problem caused by inflammatory cells or other cells
types, areas of slide that contained epithelial cells were marked with
a doting pen, and they were specifically scraped into Eppendorf tubes
for subsequent PCR analysis. From the 56 QFIA-analyzed cases, 46
yielded a sufficient amount of genomic DNA for PCR analysis. Among the
10 cases that did not have sufficient material, 4 (13.3%) were benign,
3 (16.6%) were PBDA, and 3 (12.5%) were CA; therefore, there was no
disproportion of benign versus malignant cases among those
patients who did not have sufficient material for PCR analysis. An
additional seven cases that did not undergo QFIA analysis (because only
dried smears were available) were also included in the c-myc
analysis. This study was approved by the Human Subject Protection
Committee of University of California at Los Angeles. To ensure
patients’ confidentiality, patients’ name and other identification
information were removed before the analysis.
cPCR Analysis of c-myc Oncogene Amplification.
Genomic DNA was extracted from FNA smears, many of which had undergone
QFIA studies as described above, using a slightly modified method by de
Melo et al. (8)
. Cells were first scraped into
a sterile 1.5-ml Eppendorf tube using a razor blade. Typically, from
one to two cellular smears, about 10 µg of genomic DNA was produced.
The resulting pellet was resuspended in 400 µl of 6
M guanidium hydrochloride, 30 µl of 20% sodium
sarcosyl, 30 µl of 7.5 M ammonium acetate, and
10 µl of proteinase K (10 mg/ml). Then, the entire mixture was heated
at 40°C for 1 h. An additional 10 µl of proteinase K was added
to the mixture, and the new mixture was heated for another hour at
60°C if the powder was not completely dissolved.
cPCR analysis of c-myc oncogene amplification was carried
out according to the protocol of Ribot et al.
(9)
, with minor modifications. This method
determines the amplification status of a given gene by comparing the
signal intensity of PCR product in a specimen DNA to the control DNA.
The rationale of this technique is that the signal intensity of the PCR
products reflects the copy number of the gene, and, by comparing the
signals in sample versus control DNA, the presence of gene
amplification in a specific specimen can be detected. However, instead
of using peripheral blood DNA from a normal individual as a control
source as proposed by Ribot, DNA from two cell lines were used. One
cell line (HL-60) is a high c-myc expressor, and the other
(K562) is a known low expressor for c-myc. These cells
served as positive and negative controls, respectively, for each batch
of experiments. To be noted, these cells were treated in a way
simulating the actual FNA smears (i.e., cells were
cytospined on slides, alcohol-fixed, and stained with regular
Papanicolaou before QFIA analysis).
The cPCR analysis required a calibration step using a control gene that
was not amplified in tumor DNA. In our study, GAPDH
was used as the control gene, and the up and the down primer sequences
for this gene were GCC TGC TTC ACC ACC TTC TTG and GTC CAC TGG CGT CTT
CAC CAC, respectively. For c-myc, the up and the down primer
sequences were ATG CCC CTC AAC GTT AGC TT and GTG GGC AGC TCG AAT TT,
respectively. Furthermore, the PCR amplification was performed on three
different dilutions (1:10, 1:5, and 1:2.5) of the DNA master solution
(5ng/µl). The purpose of using multiple-dilution strategy is
two-fold: (a) it served as an internal control for the
reproducibility; and (b) it kept the PCR amplification
within the exponential phase. For the calibration step, 10 µl from
each of the three dilutions of the template DNA master solution was
used to undergo PCR reaction for the control gene GAPDH. The
intensity of the bands was measured by a densitometer, and the
difference of the intensity between sample and control (HL-60 and K562)
DNA was used to calibrate the amount of diluted DNA added to the
reaction. PCR reaction using the calibrated amount of diluted DNA was
repeated for the control gene to confirm the accuracy of the
calibration. For the subsequent analysis of c-myc
amplification, the same calibrated amount of diluted template DNA was
used. The PCR cycles for both the calibration and the analysis steps
were 94°C for 40 s, 55°C for 1 min, and 72°C for 3 min for
35 cycles with 1.5 mM
MgCl2.
Data Analysis.
c-myc amplification was defined as positive if the mean DNA
band intensity in a sample was higher than the DNA band intensity in
negative control cells (K562). The degree of c-myc
amplification was further graded into +, ++, and +++ based on
densitometer measurements. 2 analysis was
carried out to determine the significance of the difference of
c-myc amplification among three study groups.
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Results
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Table 1
lists the study cases, their FNA and tissue diagnosis, and their
corresponding biomarker results. Among the 53 cases analyzed, 24 were
benign (fibroadenoma or others), 12 were PBDA, and 17 were considered
malignant or suspicious for malignancy. Twelve of the 17 cases with a
malignant diagnosis were confirmed by the subsequent tissue diagnosis.
Among the 17 cases of malignancy, there were 10 cases of infiltrating
ductal adenocarcinoma, 2 cases of intraductal papilloma with
carcinoma-in situ components, and the remaining cases were
clinically diagnosed as late-stage metastatic breast cancer.
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Table 1 List of study cases, their corresponding FNA and tissue diagnoses, the
results of c-myc amplification measured by comparative PCR,
and G-actin, p53, and DNA abnormality detected by QFIA
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Fig. 1
shows representative PCR results for c-myc amplification in
positive control cells (HL-60), negative control cells
(K562), a benign FNA sample (1-BN), and a
malignant FNA sample (2-CA). The top panel shows the PCR
products of the control gene GAPDH before calibration. The
band intensity increased as the dilution of the initial DNA added to
the reaction buffer decreased. The lower the template DNA was diluted,
the denser the band for GAPDH was after PCR amplification.
The intensity of the band densities was measured by a densitometer, and
the intrasample variation of the band intensity for each dilution was
used to calibrate the amount of template DNA added to the PCR reaction.
The middle panel shows the GADPH gene after calibration, and
the lower panel shows the results of c-myc amplification
after calibration. In this representative example, the malignant sample
(2-CA) clearly showed higher band intensity than the
negative control (K562) and the benign sample
(1-BN) and was defined as positive (+) for c-myc
amplification.
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Fig. 1. PCR analysis of c-myc in a positive control
(HL-60), a negative control (K562), a
negative sample (1-BN), and a positive sample
(2-CA).
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Table 2
summarizes the results of c-myc amplification measured by
cPCR analysis in all of the 53 samples. It was observed that 16
(94.1%) of 17 malignant lesions, 5 (41.7%) of 12 PBDA, and 4 (16.7%)
of 24 other benign lesions (fibroadenoma or fibrocystic disease) had
positive c-myc amplification. The overall difference of
c-myc amplification among these groups was highly
significant by 2 analysis (P =
0.0002).
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Table 2 c-myc amplification among three groups of breast lesions
detected by comparative PCR (P = 0.000218 by
2 test)
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Table 3
compares c-myc amplification with other biomarkers (G-actin,
p53, and DNA ploidy) among three groups of FNA samples. The
c-myc was concomitantly negative in 14 of 20 cases with
G-actin, 13 of 19 with p53, and 11 of 15 with DNA. Of about
20 cases, in which both c-myc and QFIA markers were
successfully analyzed, none of the cases were positive concomitantly
for c-myc with either G-actin or p53, except one
case with DNA ploidy. In contrast, of 14 cases in the cancer group,
c-myc was positive concomitantly in 13 cases with G-actin, 5
with p53, and 11 with DNA ploidy.
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Discussion
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The breast cancer model provides a significant opportunity
for the development of tumor markers. These tumor markers can be used
to define an individual’s risk of developing breast cancer, and they
are urgently needed in both clinical and epidemiological settings
(10)
. There are hundreds of potential tumor markers
reported in the literature; however, to determine the validity of these
markers is rather a complex process (11)
. It is well
recognized that multiple markers should be used for accurate
determination of individual risk because multiple pathways are involved
in the development of a malignant tumor (12)
. Therefore,
novel methods are needed for evaluating multiple markers simultaneously
using limited sample volume such as FNA material.
This laboratory has developed an approach that allows multiple marker
analysis to be carried out on both the phenotypic and the genotypic
level on single cell basis. The first step is to quantitatively
evaluate multiple phenotypic tumor markers (G-actin/p53/DNA)
on single-cell and in situ basis simultaneously, using a
novel QFIA method. The second step, as we reported in the current
study, is to obtain genomic DNA from the cells that had undergone QFIA
study for genotypic marker analysis such as c-myc
amplification using PCR technique. The results of these studies
indicate that by using this approach, multiple marker analysis can be
carried out even with limited cytological materials. On the basis of
these findings, it is reasonable to assume that other markers, such as
HER-2/neu and BRCA1 and 2, can also be evaluated.
Although genomic DNA has commonly been extracted from fresh or archival
tissues or recently collected cytological materials, there have been
few studies demonstrating the extraction of DNA from archival
cytologically stained and additional immunofluorescence-labeled FNA
smears. Our findings indicate that with refined technology such as
laser microdissection technique, cells with certain morphological
features or with a specific phenotype can be selected out to determine
specific molecular events underlying such a phenotype in a quantitative
fashion. This approach will have tremendous impact not only on
improving the cytological diagnosis of cancer but also on understanding
the molecular mechanisms of cancer. For example, one can compare the
expression of certain oncogenes or tumor suppressor genes between two
cells that appear normal morphologically but one of which has abnormal
phenotypes (such as increased G-actin) to determine the sequence of the
premalignant events.
This study not only shows that one can extract DNA from archival FNA
smears but also demonstrates that a semiquantitative analysis of
c-myc amplification can be carried out using the extracted
DNA material. For c-myc analysis, we used a novel cPCR
technique. Although it may seem cumbersome because it requires three
PCR reactions with different dilutions for each specimen, it is rather
simple and fast in practice. The advantage of this technique is that it
does not require an artificially introduced internal control for the
reaction. However, a potential pitfall of this method is that even with
careful measurements and volume calculations, the resulting band
intensity may still vary from the expected value. Although this problem
may be partially overcome by using three series dilutions, at best the
resulting data are only semiquantitative, and, therefore,
c-myc amplification is graded into four scales (-, +, ++,
and +++) based on the intensity reading.
Our finding that c-myc is amplified in 16.7% of benign
cases and 41.7% of PBDA cases indicates that c-myc
may not be a specific marker for cancer but probably an early risk
indicator. Some of these cases have a marked increase of
c-myc amplification (for example, case 17, Table 1
). Whether
such cases will have substantially higher risk to develop breast
malignancy is exactly the question that needs to be answered. The fact
that c-myc is positive in 4 of 18 G-actin-negative and 4 of
17 p53-negative benign lesions suggests that
c-myc amplification occurs earlier than G-actin and
p53 and, therefore, is less specific than G-actin and
p53 for breast cancer.
In summary, this study demonstrates the approach for multiple biomarker
analysis for breast cancer on FNA material. It is possible that a
similar approach can be used for other types of cytological specimens
such as urine. The information obtained and the methods developed from
this study will eventually provide a foundation for large-scale, both
retrospective and prospective, studies of biomarkers for breast cancer.
The ultimate objective of the biomarker evaluation is to develop
intermediate end-point markers for individual risk assessment that are
crucial for the success of the chemoprevention study of breast cancer.
The results of this study suggest that c-myc amplification
may be a biomarker of breast cancer risk; however, additional large,
prospective studies are needed to confirm the present observation.
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Footnotes
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 Supported in part by a seed grant from Jonsson
Comprehensive Cancer Center, awarded by National Institute of Aging,
p20 AG13095, and Grant R29-CA73108 from National Cancer Institute,
NIH. 
2 To whom requests for reprints should be
addressed, at Department of Pathology and Laboratory Medicine,
University of California-Los Angeles Medical Center, 10833 Le Conte
Avenue, Los Angeles, CA 90095-1732. Phone: (310) 794-1567; Fax: (310)
206-5178; E-mail: jrao{at}mednet.ucla.edu
3 Current address: Kaiser Permanente, Woodland
Hills Medical Center, Los Angeles, CA.
4 The abbreviations used are: FNA, fine-needle
aspiration; QFIA, quantitative fluorescence image analysis; CA,
carcinoma; PBDA, proliferative breast disease with nuclear atypia;
cPCR, comparative PCR; GAPDH, glyceraldehyde-3-phosphate
dehydrogenase.
Received 6/28/99;
revised 11/ 2/99;
accepted 11/16/99.
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Abstract
Introduction
