1. health and fitness
2. disease
3. cancer

Cancer Genetics and Cytogenetics 165 (2006) 151–156
The MYC oncogene in breast cancer progression: from benign
epithelium to invasive carcinoma
Cristina Corzoa,b,c,d,*, Josep M. Corominasc,e, Ignacio Tusquetsb,c, Marta Salidoa,
Meritxell Belletb,c, Xavier Fabregatb, Sergio Serranoe, Francesc Soléa
a
Laboratori de Citogenètica i Biologia Molecular, Servei de Patologia, Hospital del Mar, IMAS, URTTS, PRBB,
Pg. Maritim 25-29, 08003 Barcelona, Spain
b
Medical Oncology Department, Hospital del Mar, IMAS, Barcelona, Spain
c
Breast Cancer Unit, Hospital del Mar, IMAS, Barcelona, Spain
d
Departament de Biologia Cellular, Fisiologia i Immunologia, Universitat Autonòma de Barcelona, Spain
e
Pathology Department, Hospital del Mar, IMAS, URTTS, PRBB, Barcelona, Spain
Received 2 June 2005; received in revised form 3 August 2005; accepted 9 August 2005
Abstract
One hypothesis for breast cancer development suggests that breast carcinogenesis involves a progression of events leading from benign epithelium to hyperplasia (with or without atypia) to carcinoma in situ and then invasive carcinoma. The MYC gene (alias c-Myc) is a transcriptional regulator
whose expression is strongly associated with cell proliferation and cell differentiation. The present
study is a descriptive analysis of MYC status throughout the hypothesized stages of invasive ductal
carcinoma progression. A tissue microarray (TMA) was constructed including representative
selected areas (normal cells, hyperplasia, in situ carcinoma, and invasive carcinoma) from each
of 15 patients. Fluorescence in situ hybridization (FISH) with the LSI c-MYC/CEN8/IgH probe
was performed. Two cases displayed MYC amplification (13%), showing this amplification only
in the invasive carcinoma zones selected. Five cases displayed polysomy of chromosome 8
(33%), detected only in ductal in situ and invasive zones selected. Benign lesions and normal
adjacent cells were classified as normal. None of the hyperplasia specimens and normal specimens
analyzed showed any alterations in MYC status or any aneusomies of chromosome 8. The presence
of MYC amplification only in invasive cells suggests that the finding of MYC amplification could
reflect an advanced tumor progression. Ó 2006 Elsevier Inc. All rights reserved.
1. Introduction
A multistep hypothesis for breast cancer development
suggests that breast carcinogenesis involves a progression
of events leading from benign epithelium through to hyperplasia (with or without atypia) to in situ carcinoma and
invasive carcinoma [1].
It is known that the finding of in situ carcinoma increases by 20–25 times the risk of developing invasive
breast cancer. Although associations are evident between
some forms of benign breast disease and the increased risk
of malignancy, confirmatory evidence that the benign breast
disease lesions are biologically premalignant has not yet
been established [2].
* Corresponding author. Tel.: 134-93-2483035; fax: 134-93-2483131.
E-mail address: E0062@imas.imim.es (C. Corzo).
0165-4608/06/$ – see front matter Ó 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.cancergencyto.2005.08.013
Complex and heterogeneous sets of genetic alterations
are involved in the etiology of breast cancer. It is believed
that breast cancer has its origin in a single cell that, through
a number of different events, becomes malignant. Theory
explains tumor development as the result of clonal selection
and evolution within a cell population that accumulates
a set of genetic aberrations. Additional events lead to the
development of different clones with different features
[3,4]. Although the sequential steps in gene alteration, with
respect to breast tumor progression, are poorly understood,
in instances involving the breast these have been proven for
invasive breast cancer and for premalignant lesions, such as
ductal carcinoma in situ. In contrast, a polyclonal expansion is expected in hyperplastic lesions, in which cells of
multiple origin respond to exogenous and endogenous
stimuli [5]. The evolution from one pathological lesion to
another has been associated with different genetic events,
such as the activation of oncogenes and the inactivation
of tumor suppressor genes. It seems that gene amplification
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C. Corzo et al. / Cancer Genetics and Cytogenetics 165 (2006) 151–156
is a late event in tumor progression, being found mainly in
tumor cells that have acquired genetic instability and
tolerate its presence. Nevertheless, few studies confirm this
hypothesis [6].
The c-myc oncoprotein encoded by the MYC gene is
a transcriptional regulator whose expression is strongly associated with cell proliferation and cell differentiation [7].
MYC is implicated in most cellular functions, such as replication, growth, metabolism, differentiation, and apoptosis
[8]. The incidence of MYC amplification has been studied
in several tumors (e.g., bladder, lung, and head and neck tumors) having different percentages of positivity [9]. In
breast carcinoma, the incidence of MYC amplification
ranges from 1 to 94% in different studies [10–13].
The present study is a descriptive analysis of MYC status
throughout the hypothesized stages of invasive ductal carcinoma progression, in order to detect a clonal origin during
the evolution of breast cancer. To our knowledge, the present study is the first that compares MYC status in different
histopathological regions identified in the same patients,
starting from benign epithelium and preinvasive lesions
and following through to invasive cancer. This particular
study was made possible using tissue microarray (TMA)
technology to obtain representative areas of each sample.
TMA technology makes it possible to analyze a high number of different specimens in a single assay, therefore decreasing the variability of results among specimens
treated in different assays. In our study, fluorescence in situ
hybridization (FISH) in TMA paraffin block was used to
evaluate MYC status throughout the multiple steps of breast
cancer progression, using a homogeneous series of invasive
ductal carcinomas and excluding lobular carcinomas or
other histologies.
2. Materials and methods
Twenty cases were selected from a cohort of patients
who underwent therapeutic surgery for a first incidence of
breast cancer. This group of patients presented four different areas upon histopathological examination: normal cell
pathology, hyperplasia without atypia, in situ carcinoma,
and invasive carcinoma. The clinical and pathological data
of this cohort are summarized in Table 1.
The collection of biological specimens was obtained
from the tissue bank at the Hospital del Mar (IMAS, Barcelona, Spain). Approval was obtained by the ethical committees at the Hospital del Mar. Tissue bank informed consent
was provided, according to the Declaration of Helsinki.
Samples were fixed with a 4% buffered formalin and
embedded in paraffin. Sections of the paraffin-embedded
tissue (4–6 mm thick) were mounted in silanized slides
and either stained with hematoxylin–eosin (H&E) or tested
using immunohistochemical methods for hormonal receptors, TP53, and ERBB2 and using FISH for ERBB2.
The H&E-stained sections from tumor samples included
in paraffin blocks were used to identify the different zones
Table 1
Baseline clinical and pathological characteristics of 15 cases
of invasive ductal carcinoma
Characteristic
Stage
I
II
IIIa
Histological grade
I
II
IIIa
Hormone receptor status
ER1, PR1
ER1, PR2
ER2, PR1
ER2, PR2
TP53
1
2
ERBB2
31 (FISH1)
21
Negative (01, 11)
No.
%
7
6
2
47
40
13
6
8
1
40
53
7
8
4
1
2
53
27
7
13
3
12
20
80
3
0
12
20
0
80
Abbreviations: ER, estrogen receptor; PR, progesterone receptor.
Median age: 59.8 years (range 43–79).
of tumoral cells (proliferative lesions, in situ carcinoma,
and infiltrating carcinoma) and also normal cells, taken
from areas around the tumors. We were then able to construct a TMA as described by Kononen et al. [14] using
a commercially available TMA system (Beecher Instruments, Sun Prairie, WI). We obtained a TMA with 160
spots (1 mm in diameter, each); the array included two
spots of each lesion from every patient. Six tissue controls
were included to assess the standard values of FISH. Consecutive sections from the TMA were obtained for H&E
staining and for FISH analysis. An expert pathologist performed a histological check to confirm that TMA array contained the spots with proliferative lesions, in situ carcinoma
and infiltrating carcinoma and also normal cells from each
patient selected (Fig. 1).
To perform FISH, the LSI c-MYC/CEN8/IgH probe
(Vysis, Downers Grove, IL) was used: the MYC-specific locus probe was labeled in orange, centromere 8 in aqua, and
IgH in green. The IgH probe is used to detect translocations
in hematological malignancies; in this case, the IgH probe
was not evaluated.
Deparaffinized TMA tissue sections were treated with
0.2 mol/L hydrochloric acid and then with sodium thiocyanate to eliminate salt precipitates. Pretreated slides were
incubated in a proteinase K solution for 10 minutes at
37 C to digest the proteins of cytoplasmic membrane. Then
the slides were postfixed in buffered formalin.
Pretreated tissue sections and probes were denatured at
78 C for 5 minutes and hybridized overnight at 37 C in
a Hybrite chamber (Vysis). Three washes for 10 minutes
each were performed at 45 C in a formamide 50% solution,
and two washes at the same temperature in 2 saline
C. Corzo et al. / Cancer Genetics and Cytogenetics 165 (2006) 151–156
Invasive carcinoma cells
In situ carcinoma
Hyperplasia
Normal
153
Controls
Fig. 1. Scheme of the spots included in the tissue microarray that contained all lesions for each of the 15 patients selected.
sodium citrate solution for 5 minutes each. Tissue sections
were counterstained with 10 mL of 40 ,6-diamidino-2phenylindole (DAPI) (Vysis).
Results were analyzed in a fluorescent microscope
(Olympus BX51), using Cytovision software (Applied Imaging, Newcastle upon Tyne, UK). Tissue sections were
scanned at low magnification (100) with DAPI excitation
to localize those areas in which the histopathological characteristics had been established through examination of the
serially sectioned H&E-stained stained array sections.
Eight spots from tissue controls were scored, to establish
a cutoff to accurately define true amplifications and aneusomies. These controls were from normal nonbreast tissues.
Standards were set so that cells with three signals of MYC,
with respect to two signals of centromere 8, in O10% of
the nuclei were considered to be a gain. Fewer than two signals in O50% of the nuclei were considered to indicate
a loss. A ratio between MYC signals and centromere signals
was established, and amplification was considered when
this ratio was >2. These results are consistent with previously reported results [15]. We considered polysomy of
chromosome 8, when >3 signals of centromere 8 per cell
were observed.
When possible, 100 nuclei were scored for each spot;
however, in spots corresponding to benign cells, the number
of cells available was sometimes smaller than for in situ or
invasive lesions.
digestion of some tissues, and weak hybridization. An important technical problem when analyzing a TMA is that
smaller samples are sometimes lost from the slides during
the technical procedure. It is important to consider that
the pretreatment procedure needed for each sample can
vary, but in a TMA it is not possible to perform different
digestion for each tissue. This is the reason that some samples displayed a weak hybridization.
We found two cases with MYC amplification (13%),
with the amplification seen only in the invasive zones; the
in situ carcinoma, hyperplasia, and normal cells from these
two patients did not display MYC amplification and these
cells were classified as normal. One case displayed three
copies of the MYC gene, with respect to two centromeres,
in 14% of cells; this was classified as a gain, but not as
an amplification. This gain was found only in invasive
and in situ cells. Five cases displayed polysomy of chromosome 8 (33%), detected only in ductal in situ and invasive
zones; the hyperplastic and normal cells did not show any
alteration. All cases were classified as normal in terms of
evaluation of benign lesions and normal adjacent cells.
To describe the relation of MYC status with histology
grade, we observed that amplified cases displayed a grade
II in the in situ component and also in invasive cells. Polysomic cases displayed different histology grades. Table 2
summarizes the FISH results from the present study, and
Table 3 summarizes the correlation between FISH results
and histological data.
3. Results
Twenty cases were included in the TMA, but only 15
cases were available for FISH analysis of MYC status,
due to the loss of 5 samples during the manipulation of
TMA or due to unsuccessful hybridization. Reasons for unsuccessful analyses included tissue damage, incorrect
4. Discussion
MYC is an oncogene with a central role in tumor progression, and several studies have attempted to clarify its
role in carcinogenesis [16–18]. Previous reports have
C. Corzo et al. / Cancer Genetics and Cytogenetics 165 (2006) 151–156
154
Table 2
Results found after FISH analysis with a c-MYC probe from specimens
of the four different areas included in the tissue microarray
Case no.
Normal
cells
Hyperplasia
In situ
carcinoma
Invasive
carcinoma
1
2
3
4
5
6
7
8
9–15
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Polysomic
Polysomic
Polysomic
Polysomic
Polysomic
Gain
Normal
Amplified
Amplified
Polysomic
Polysomic
Polysomic
Polysomic
Polysomic
Gain
Normal
analyzed MYC status in breast cancer patients; most of
them studied this oncogene in invasive carcinoma, but none
included normal cells, benign lesions, and preinvasive
carcinomas from the same patient. Although our series of
patients was small, to our knowledge the present study is
the first that has evaluated different areas of the same breast
tumor specimen (normal cells, hyperplasia without atypia,
in situ carcinoma, and invasive carcinoma) using TMA
technology, in order to describe the oncogenetic involvement of MYC in breast cancer progression.
In our series, MYC amplification was present in 2 cases
out of 15 (13%). Within the group of amplified cases, only
invasive cells displayed MYC amplification. The other areas
studied from the same tumor (normal cells, hyperplasia
without atypia, and in situ carcinoma) were classified as
normal. This finding may indicate that MYC amplification
is present only in those cells that have acquired a malignant
phenotype.
Several studies have found similar percentages of amplification, including those conducted by Rummukainen et al.
[19] and Robanus-Maandag et al. [20]. The latter study
compared MYC status between invasive and in situ components, and their results showed amplification in invasive
cells only, whereas in situ components were always normal
[20]. These results are in agreement with our own findings
and suggest that MYC amplification may be associated with
the progression from in situ carcinoma to the invasive stage
of ductal carcinoma of the breast.
Aulmann et al. [21] studied MYC amplification in in situ
carcinoma of the breast; they found that ~20% of their
cases showed amplification of the MYC oncogene, mostly
in poorly and intermediately differentiated tumors. They
concluded that the MYC oncogene plays a role in the pathogenesis of a subset of in situ ductal carcinomas having an
unfavorable tumor biology. Furthermore, it is likely that invasive recurrences of these intraductal cancers may lead to
MYC amplification, resulting in tumors with a poor prognosis. This study may seem to be contradictory to our own results, but not if we consider that the MYC amplification
found by Aulmann et al. [21] in the in situ carcinoma
was present only in the more aggressive cases.
In a recently published study, Blancato et al. [22] reported
a high level of MYC amplification (70%). This experience included only high-grade invasive breast cancers; we believe
that this is probably the reason for the discrepancy. Table 4
summarizes MYC analyses reported by different authors.
Chromosome 8 polysomy is a frequent alteration in
breast tumors, although the prognostic value of this aberration is still uncertain [23–25]. In our series, polysomy of
chromosome 8, present in 33% of the cases analyzed,
was found in both in situ carcinoma and invasive cells from
the same patients. Benign proliferative lesions and normal
cells, taken from the tumor margins, did not present MYC
amplification or chromosome 8 aneusomies. In our study,
the finding of polysomy 8 in carcinomas only (i.e., in situ
and invasive cells) suggests that this is an aberration, solely
present in true tumors, but not present in premalignant lesions. These results are consistent with previously reported
data [23–29].
Bofin et al. [28] studied the polysomy of chromosome 8
and found that 53% of the breast tumor cases analyzed displayed polysomy of this chromosome; out of these cases,
89% were classified as intermediate or high-grade invasive
carcinomas. Tagawa et al. [29] and Fehm et al. [27] in their
series published similar results. Visscher et al. [12] studied
MYC amplification and chromosome 8 aneusomies in
Table 4
Review of MYC amplification in previous reports
References
Table 3
Histological data for the amplified and polysomic cases
Case no.
FISH
Grade
ER
PR
TP53
ERBB2
1
2
3
4
5
6
7
8
Amplified
Amplified
Polysomic
Polysomic
Polysomic
Polysomic
Polysomic
Gain
2
2
1
1
3
2
2
1
2
1
2
1
1
1
1
1
2
1
2
1
1
2
1
1
1
2
1
2
2
2
2
2
1
2
1
2
2
2
2
2
The most representative pathological events are included in this table,
but any statistical conclusion may be assessed.
No. of Invasive
cases cells, %
Schraml et al., 1999 [9]
68
Visscher et al., 1997 [12] 33
Rummukainen et al.,
261
2001 [13]
Selim et al., 2002 [15]
48
Rummukainen et al.,
177
2001 [19]
Robanus-Maandag et al., 188
2003 [20]
Aulmann et al., 2002 [21] 96
Blancato et al., 2004 [22] 46
a
b
c
23
91a
14.6
In situ
cells, %
Benign
lesions, %
17
Not studied
100b
0
Not studied Not studied
Not studied Not studied 0
14
Not studied Not studied
9.6
0
Not studied
Not studied 20
Not studied
70
Not studied Not studiedc
Includes both low-level and high-level amplification.
A single case.
Used as control.
C. Corzo et al. / Cancer Genetics and Cytogenetics 165 (2006) 151–156
a series of 23 invasive breast carcinomas, 1 low-grade in
situ ductal carcinoma, 7 benign lesions, and 2 phyllodes tumors. They found that neither the benign lesions nor the in
situ carcinoma or phyllodes tumors showed MYC amplification or chromosome 8 polysomy. Nevertheless, 90% of the
carcinomas showed some of these alterations. These percentages of alterations (polysomy and amplification) were
higher than the percentages we found, but this fact may
be due to the different cutoff levels used and to the smaller
series in our study.
It is also necessary to consider that we studied a cohort
of patients that presented different histological regions
within their tumoral lesions. This group of patients may
not necessarily bear the same genetic alterations that other
tumors in which histologically different lesions are not
detected.
In conclusion, to our knowledge the present study is the
first that compares MYC status in different lesions from the
same patient, in order to study breast cancer progression;
nevertheless, the number of patients analyzed is too low
to establish a conclusion. The results found in the present
study reveal MYC amplification as an unusual event in ductal breast carcinoma; the results could also suggest that
MYC amplification appears in the final stages of breast cancer development. None of the hyperplasia specimens and
normal specimens analyzed showed any alterations in
MYC status or any aneusomies of chromosome 8. The presence of MYC amplification only in invasive cells may indicate that the finding of MYC amplification could reflect an
advanced tumor progression and it could be a method used
in cytology to differentiate between preinvasive lesions and
true invasive lesions in some difficult cases. The detection
of MYC status by FISH in patients with breast cancer could
have prognostic value, due to its implication in the
advanced stages of tumor development.
Acknowledgments
We want to thank Beatriz Bellosillo for the review of the
manuscript. This work was supported by grant no. FIS 02/
0002 from the ‘‘Ministerio de Sanidad y Consumo,’’ by
grant no. SAF-2001–4947 from the ‘‘Ministerio de Ciencia
y Tecnologı́a,’’ and by grants from Redes de Centros de Genética, no. C03/07, and Redes de Cáncer, no. C03/10, from
the Instituto de Salud Carlos III from the ‘‘Ministerio de
Sanidad y Consumo,’’ Spain.
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