Human Pathology (2012) 43, 4855

www.elsevier.com/locate/humpath

Original contribution

The hormone receptor, human epidermal growth factor

receptor 2, and molecular subtype status of individual
tumor foci in multifocal/multicentric invasive ductal
carcinoma of breast
Yoomi Choi MD a , Eun Joo Kim b , Hyesil Seol MD b , Hee Eun Lee MD, PhD b ,
Mi Jung Jang MD c , Sun Mi Kim MD, PhD c , Jee Hyun Kim MD, PhD d ,
Sung-Won Kim MD, PhD e , Gheeyoung Choe MD, PhD a,b , So Yeon Park MD, PhD a,b,
a

Department of Pathology, Seoul National University College of Medicine, Seoul, 110-799, Korea
Department of Pathology, Seoul National University Bundang Hospital, Seongnam, Gyeonggi, 463-707, Korea
c
Department of Radiology, Seoul National University Bundang Hospital, Seongnam, Gyeonggi, 463-707, Korea
d
Department of Internal Medicine, Seoul National University Bundang Hospital, Seongnam, Gyeonggi, 463-707, Korea
e
Department of Surgery, Seoul National University Bundang Hospital, Seongnam, Gyeonggi, 463-707, Korea
b

Received 27 April 2010; revised 5 August 2010; accepted 20 August 2010

Keywords:
Breast cancer;
HER-2;
Hormone receptor

Summary Multifocal/multicentric breast cancers are common. However, investigations of biomarkers

such as estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 in
individual tumor foci of such cancers are rare. This study was designed to evaluate the status of the
hormone receptors, human epidermal growth factor receptor 2, and its molecular subtypes in individual
foci of multifocal/multicentric invasive ductal carcinoma of the breast and to identify the factors
associated with the different phenotypes of individual foci. We performed immunohistochemical
analyses of the estrogen receptor, progesterone receptor, cytokeratin 5/6, epidermal growth factor
receptor, and p53 and fluorescence in situ hybridization of human epidermal growth factor receptor 2 in
individual foci of 65 cases of multifocal/multicentric invasive ductal carcinoma and the associated
ductal carcinoma in situ components using tissue microarrays. The estrogen receptor status differed in 2
(3%) of the 65 invasive ductal carcinomas, progesterone receptor status in 7 (11%), human epidermal
growth factor receptor 2 status in 4 (6%), and molecular subtypes in 5 (8%). The presence of different
molecular subtypes in the invasive tumor foci was associated with differences in histologic features (P =
.005), high histologic and nuclear grade (P = .012 and P = .021, respectively), p53 overexpression (P =
.006), and mixed molecular subtypes in the ductal carcinoma in situ components (P = .011). Multifocal/
multicentric invasive ductal carcinomas usually have a single phenotype in terms of hormone receptors,
human epidermal growth factor receptor 2, and molecular subtypes; and thus, immunohistochemical
analyses of the index tumor may be sufficient in routine practice. However, if multifocal/multicentric

Grant support: This study was supported by a grant from the Seoul National University Bundang Hospital, Seongnam, Korea (Research Fund Grant
No. 03-2009-01) to S. Y. Park.
Corresponding author.
E-mail address: sypmd@snu.ac.kr (S. Y. Park).

0046-8177/$ see front matter 2012 Elsevier Inc. All rights reserved.
doi:10.1016/j.humpath.2010.08.026

Hormone receptor, HER-2, and molecular subtype status

49

invasive ductal carcinomas are of high grade, of different histologic features, or of heterogeneous ductal
carcinoma in situ component, biomarkers of the various foci need to be evaluated separately.
2012 Elsevier Inc. All rights reserved.

1. Introduction
Breast cancers can present as multifocal or multicentric
tumors. Estimates of the frequency of such tumors differ
depending on the histologic methods used, the diagnostic
criteria applied, and case selection, ranging from 6% to
75% [1-7]. Although there is no international consensus on
the definition of multicentricity or multifocality, multicentricity generally is said to be the presence of at least one
clinically or mammographically evident tumor in a different
breast quadrant from the index lesion; and multifocality is
the presence of more than one distinct focus in a given
quadrant [2].
The development of multifocal/multicentric cancer in the
same breast could be explained by 2 mechanisms. First, it
could arise as multiple independent tumors. In that case, the
lesions could have different phenotypes and underlying
molecular changes. Second, it could reflect intramammary
spread of a tumor. In fact, all tumor foci are thought to have
the same phenotype, although genetic or phenotypic
alterations can occur during tumor progression. According
to the current American Joint Committee on Cancer manual
(seventh edition), T stage is determined by the size of the
largest invasive tumor focus in a multifocal/multicentric
cancer [8]. Thus, the other tumor foci usually are not
considered important in selecting further treatment. Immunohistochemical analyses of biomarkers, including estrogen
receptor (ER), progesterone receptor (PR), and human
epidermal growth factor receptor 2 (HER-2), are performed
either on the index tumor or on each separate invasive tumor.
If one examines only the index tumor, one would miss any
tumors that have different immunophenotypes and might
thus need different systemic treatment from the index tumor.
In a previous study that evaluated the ER and PR status of
individual foci in multifocal breast cancer, the ER status was
the same for each focus in all 18 cases [9]. In another study,
ER, PR, and HER-2 status was the same in the separate
tumors of 32 multicentric breast cancers; and the authors
suggested that the results supported the practice of evaluating
prognostic markers in only one lesion per cancer [10].
However, these studies involved only small numbers of
cases; and none evaluated the molecular subtypes of the
individual foci of multifocal/multicentric breast cancers.
In this study, we evaluated the histologic features, ER,
PR, HER-2, and molecular subtypes of individual tumor
foci in 65 cases of multifocal/multicentric invasive ductal
carcinoma (IDC) of the breast to determine if all the tumors
in individual patients have the same characteristics and thus
whether it is sufficient to examine the index tumor alone

for the purpose of immunohistochemical analysis. We also

tried to find the characteristics of multifocal/multicentric
IDCs with discordant immunohistochemical features in
individual foci.

2. Materials and methods

2.1. Tissue specimens
From January 2006 to May 2009, 485 patients underwent
surgical excision of the breast for IDC at Seoul National
University Bundang Hospital, of which 110 (23%) were
found to have multifocal/multicentric IDC. Of these 110
patients, 45 were excluded from the study because of a
history of preoperative systemic chemotherapy or insufficient evidence of multifocality from a review of slides. In this
study, multicentricity was defined as the presence of a
radiologically/grossly evident and histologically confirmed
tumor in a different breast quadrant from the index lesion;
and multifocality was defined as the presence of more than
one distinct focus in a given quadrant based on radiologic
and histologic findings. When invasive cancers were grossly
and radiologically separate and histologic examination
revealed intervening nontumorous tissue (N5 mm), the
lesions were regarded as multifocal tumors. When grossly
or radiologically distinct tumors were very close (b5 mm),
especially if they had similar histologic features, they were
regarded as the same tumor. In the end, 65 cases of
multifocal/multicentric IDCs, composed of 54 multifocal and
11 multicentric cancers, were included in this study. Fortytwo patients had 2 separate invasive tumor foci, 12 had 3, 5
had 4, 5 had 5, and the last had 6. All tumors were IDC, not
otherwise specified, although one case showed a minor
mucinous component.
After review of the slides, all invasive tumor foci in each of
the 65 multifocal/multicentric breast cancers were selected.
Of the 65 tumors, 61 (94%) had a ductal carcinoma in situ
(DCIS) component in at least one of the multiple tumor foci.
Forty-seven cases had a sufficient DCIS component associated with the 2 largest invasive tumor foci for evaluation, and
the DCIS components were selected for study.
Tissue microarrays (TMAs) of 4-mm cores were
constructed from the invasive tumor foci in the 65 IDCs
and from the DCIS component in 47 cases using a trephine
apparatus (Superbiochips Laboratories, Seoul, Korea). All
the patients were female with a mean age of 48.5 11.2 (SD)
years (range, 21-75 years). Clinicopathologic information
was obtained by reviewing pathology reports and

50
hematoxylin and eosin (H&E)stained sections. The following histopathologic variables were determined for each
tumor focus: size, histologic subtype, modified Black
nuclear grade [11], Nottingham combined histologic grade
(tubule formation, nuclear pleomorphism, mitotic count),
presence or absence of an intraductal component, lymphatic
and venous invasion, and tumor border. Histologic similarities among the invasive tumor foci were determined on the
basis of the modified Black nuclear grade, growth pattern
(solid, tubular, cribriform, cordlike, etc), and stromal
component (no stroma, desmoplastic, inflammatory, etc).
For the associated DCIS components, we recorded nuclear
grade, architectural pattern, and presence of necrosis. All
cases were independently reviewed by 2 breast pathologists
(S. Y. P. and H. S.). This study was approved by the
Institutional Review Board of Seoul National University
Bundang Hospital.

2.2. Immunohistochemical analyses

Four-micrometerthick TMA sections were cut, dried,
deparaffinized, and rehydrated following standard procedures. All the sections were subjected to heat-induced
antigen retrieval in citrate buffer (pH 6.0). Immunohistochemical staining was carried out in a DAKO Autostainer
Plus (Dako, Glostrup, Denmark) using an LSAB detection
kit Dako. The following mouse monoclonal antibodies were
used: ER (clone SP1, 1:100, Labvision, Fremont, CA), PR
(clone PgR 636, 1:70, DAKO, Carpinteria, CA), HER-2
(polyclonal, 1:700, DAKO), p53 (clone DO-7, 1:600,
DAKO), cytokeratin 5/6 (clone D5/16 B4, 1:50, DAKO),
and epidermal growth factor receptor (EGFR) (EGFR
pharmDx, DAKO).

2.3. Fluorescence in situ hybridization assays for

HER-2 gene amplification
To determine HER-2 gene amplification, PathVysion
(Vysis, Downers Grove, IL) assays were carried out and
evaluated as previously described [12]. At least 50 cells
were evaluated for each TMA core; and the HER-2 gene
was considered to be amplified in tumors with a ratio of
HER-2 to CEP17 of at least 2.2 according to American
Society of Clinical Oncology/College of American Pathologists guideline [13].

2.4. Definition of breast tumor subtypes

Breast tumor subtypes were defined as previously [14,15]
with minor modifications. Subtype definitions were as
follows: luminal A (ER+ and/or PR+, HER-2), luminal B
(ER+ and/or PR+, HER-2+), HER-2+ (ER, PR, HER-2+),
basal-like (ER, PR, HER-2, cytokeratin 5/6+, and/or
EGFR+), and unclassified (negative for all markers). HER-2
positivity was determined from the fluorescence in situ
hybridization (FISH) results.

Y. Choi et al.

2.5. Immunohistochemical scoring

Expression of EGFR and HER-2 was scored as follows: 0,
no staining; 1+, weak and incomplete membranous staining
in at least 10% of the tumor cells; 2+, weak to moderate,
complete membranous staining in at least 10% of the tumor
cells; and 3+, strong, complete membranous staining in at
least 30% of the tumor cells. Any positive staining was
regarded as positive for EGFR, and 3+ was considered
positive for HER-2. Both ER and PR were scored using the
Allred system [16]. The proportion scores, which represent
the estimated proportion of positive tumor cells (0, none; 1,
b1/100; 2, 1/100 to 1/10; 3, 1/10 to 1/3; 4, 1/3 to 2/3; 5, N2/3)
and intensity scores (0, none; 1, weak; 2, intermediate; and 3,
strong), were added to obtain a total score, which ranged
from 0 to 8. A total score of 3 or more was regarded as
positive. For p53, cases with 10% or more positive staining
were grouped as positive. For cytokeratin 5/6, cases with any
degree of positive staining were considered positive.

2.6. Statistical analysis

Statistical analysis was performed with SPSS software
(version 12.0; SPSS Inc, Chicago, IL). Fisher exact test was
used when comparing frequencies between groups. All
numerical data were expressed as means standard deviation.
A P value b .05 was considered statistically significant.

3. Results
3.1. Histologic findings
The nuclear or histologic grade and histologic similarity
of each invasive carcinoma were assessed from the whole
sections. Nuclear or histologic grade was the same in 57
cases (88%), and the histologic pattern was the same in 41
cases (63%). The nuclear grades of the invasive and DCIS
components were also evaluated in 94 tumor foci (2 largest
foci in each of the 47 multifocal/multicentric IDCs with a
DCIS component), and they were the same in 86 (91%). Of
the 8 tumor foci with different nuclear grades in the invasive
and DCIS components, 7 were of mixed nuclear grade in the
DCIS component.

3.2. Comparison of hormone receptor, HER-2,

and molecular subtypes in the invasive tumor foci
The ER status was the same for the invasive tumor foci in
63 (97%) of the 65 multifocal/multicentric IDCs. ERs were
uniformly present in 47 cases (72%), completely negative in
16 cases (25%), and different in 2 cases (3%). The PR status
of the individual tumor foci was positive in 38 (58%) and
negative in 20 (31%) cases. Of the 7 cases (11%) where the
PR status differed, 6 were ER positive; and in the remaining

Hormone receptor, HER-2, and molecular subtype status

51

case, the ER status differed. Amplification of HER-2 was

detected in all tumor foci in 16 cases (25%). In 4 cases (6%),
the HER-2 amplification status was different.
Molecular subtype was defined by the status of hormone
receptors, HER-2 amplification, and expression of CK5/6
and EGFR. Sixty cases (92%) had the same molecular
subtypes, with 39 luminal A, 6 luminal B, 9 HER-2+, and 6
basal-like subtypes. Molecular subtype differed among the
individual tumor foci in 5 cases (8%): luminal A (first tumor)
and luminal B (second tumor) in 1 case, basal-like (first,
second, third, and fifth tumor) and HER-2+ subtype (fourth
tumor) in 1 case, HER-2+ (first tumor) and luminal B
subtype (second tumor) in 1 case, luminal B (first tumor) and
luminal A subtype (second and third tumor) in 1 case, and
luminal A (first tumor) and HER-2+ subtype (second tumor)
in the remaining case (Table 1; Figs. 1 and 2).

tumor (P = .021 and P = .012, respectively). Four (80%) of

the 5 cases had p53 overexpression, compared with 10 (17%)
of the 60 cases with the same molecular subtypes (P = .006).
Three (60%) of the 5 cases had mixed molecular subtypes in
the DCIS component, whereas this was true of only 3 (7%) of
the 42 cases with the same molecular subtype (P = .011)
(Table 2).

3.3. Characteristics of the multifocal/multicentric

IDCs with different molecular subtypes
The 5 cases with different molecular subtypes in the
invasive tumor foci were all multifocal. All the cases showed
different histologic features among individual tumor foci,
whereas only 19 of 60 cases with the same molecular
subtypes had different histologic features (P = .005). All 5
cases had high nuclear and histologic grade in the index
Table 1

4. Discussion
Multifocal/multicentric breast cancers are common, but
their origin is not clear. Multiple foci could be the result of
spreading of a single primary tumor or, alternatively, of
tumors arising independently in separate progenitor cells.
Some methods of distinguishing tumor origins have been
reported. Previous studies evaluated histologic and immunohistochemical features [10,17] and found that most
multicentric breast cancers had similar histologic and
immunohistochemical characteristics. Middleton et al [10]
therefore suggest that early-stage synchronous tumors derive
from a single mammary carcinoma. However, in our study,
37% of multifocal/multicentric breast cancers had different
histologic features; and 8% contained different molecular
subtypes. Dawson et al [17] also reported that 9 (38%) of 24
multifocal breast cancers had differing histologic features

Multifocal/multicentric breast cancer with different phenotype in the individual tumor foci

Case no.

Component

Size (cm)

Histology

HG

NG

ER a

PR a

HER-2 b

Subtype

1st IDC
1st DCIS
2nd IDC
2nd DCIS
1st IDC
1st DCIS
2nd IDC
2nd DCIS
3rd IDC
4th IDC
5th IDC
1st IDC
1st DCIS
2nd IDC
2nd DCIS
1st IDC
1st DCIS
2nd IDC
2nd DCIS
3rd IDC
1st IDC
1st DCIS
2nd IDC
2nd DCIS

0.7

Different

3
3
3
3&2
3
3
3
3
3
3
3
3
3
3
3
3
3
1
1&3
1
3
3
3
3

7
6/0
6
8
0
0
0
0
0
0
0
0
0
5
7/0
4
8
7
8
7
4
6
0
0

6
5/0
5
7
0
0
0
0
0
0
0
0
0
1
7/0
8
8
7
8
7
0
0
0
0

0
1
1
1/0
0
0
0
0
0
1
0
1
1
1
1
1
1
0
0/1
0
0
0
1
1

Luminal A
Luminal B/HER-2+
Luminal B
Luminal B/luminal A
Basal-like
Basal-like
Basal-like
Basal-like
Basal-like
HER-2+
Basal-like
HER-2+
HER-2+
Luminal B
Luminal B/HER-2+
Luminal B
Luminal B
Luminal A
Luminal A/luminal B
Luminal A
Luminal A
Luminal A
HER-2+
HER-2+

44

56

63

65

0.5
2.5

3
Different

1.0

0.7
0.7
0.3
1.0

3
3
3
3

Different

1.0
1.5

3
Different

1.4
1.0
1.8
1.5

3
1

Different

1
3
3

Abbreviations: NG, nuclear grade; HG, histologic grade.

a
ER and PR were scored using the Allred scoring system.
b
HER-2 status was evaluated by FISH: 1 (amplified); 0 (not amplified).

52

Y. Choi et al.

Fig. 1 Mixed molecular subtypes in the DCIS component. A representative example (case 56) of multifocal/multicentric IDC of the breast
with mixed molecular subtypes in DCIS component. The invasive and DCIS component of the first tumor is ER negative, PR negative, and
HER-2 positive (HER-2+ subtype). However, the invasive component of the second tumor is ER positive, PR positive, and HER-2 positive
(luminal B subtype), whereas the DCIS component is ER positive/negative, PR positive/negative, and HER-2 positive (mixed luminal B and
HER-2+). Magnification: 200 (H&E, immunohistochemical staining) and 1000 (FISH).

and that 12 (50%) of them yielded different immunohistochemical staining patterns. However, we cannot be sure that
multifocal/multicentric breast cancers of different phenotype
are of independent origin because phenotypic changes can
occur during tumor progression and dissemination [18,19].
Molecular genetic analyses have been performed to
determine the origin of multifocal/multicentric breast
cancers. Tsuda and Hirohashi [20] examined loss of
heterozygosity on chromosome 16q in multiple breast
cancers and concluded that multicentric cancers defined as
those not connected via the DCIS component and not
showing satellite nodules could arise independently. On the
other hand, Teixera et al [21,22], using cytogenetic analysis,
concluded that the dominant origin of multiple breast cancers
is intramammary spread from a single primary tumor,
although some cases arise by independent pathogenic
processes. Recently, Brommesson et al [23] compared
genomic similarities among synchronous multiple invasive
breast cancers by microarray-based comparative genomic
hybridization and found that 5 of 10 unilateral tumor pairs
displayed similar genomic profiles, suggesting that some
synchronous unilateral multiple tumors can have a common
origin, whereas others arise independently.
Hormone receptor and HER-2 status of individual tumor
foci in multifocal/multicentric breast cancers may have
clinical implications. Immunohistochemical assessment of

ER and PR status is standard practice in patients with breast

cancer to identify those who need hormone therapy, and
assessment of HER-2 status by immunohistochemistry or
FISH is also used to identify patients who are most likely to
benefit from trastuzumab [24,25]. In our study, 5 (8%) of 65
multifocal/multicentric breast cancers had different phenotypes. Thus, if we examined only the index tumor in such
cases, we might miss patients who needed trastuzumab or
hormone therapy. Interestingly, one patient with the luminal
A and HER-2+ subtype also had different subtypes in the
lymph node metastases: 2 lymph nodes with luminal A
subtype and 1 with the HER-2+ subtype. Compared with
IDCs, invasive lobular carcinomas (ILCs) are ER and PR
positive at a higher rate and show HER-2 overexpression at a
lower rate with the exception of pleomorphic lobular
carcinoma [26]. Thus, multifocal/multicentric ILCs are likely
to have the same immunohistochemical features in hormone
receptors and HER-2 expression. If we combined multifocal/
multicentric ILCs, there may be a greater concordance of
immunohistochemical results. Differences in the molecular
subtypes of invasive tumor foci in multifocal/multicentric
IDCs were associated with different histologic features of the
tumors (P = .005), high histologic or nuclear grade (P = .012
and P = .021, respectively), p53 overexpression (P = .006),
and mixed molecular subtypes in the DCIS components (P =
.011).Thus, if multifocal/multicentric IDCs are of high grade,

Hormone receptor, HER-2, and molecular subtype status

53

Fig. 2 Different molecular subtypes in individual tumor foci of multifocal/multicentric breast cancers. Representative example (case 65) of
multifocal/multicentric IDC of the breast with different molecular subtypes in individual tumor foci. The invasive and DCIS components of the
first tumor are ER positive, PR negative, and HER-2 negative (luminal A subtype), whereas the invasive and DCIS components of the second
tumor are ER negative, PR negative, and HER-2 positive (HER-2+ subtype). Magnification: 200 (H&E, immunohistochemical staining) and
1000 (FISH).

of different histologic features, or of heterogeneous DCIS

component, biomarkers of the multiple foci may need to be
evaluated separately.

Table 2

Our study showed that a difference in molecular subtypes

among the invasive tumor foci was associated with mixed or
heterogeneous molecular subtypes of the DCIS components.

Characteristics of multifocal/multicentric breast cancers with different subtypes in the individual tumor foci

Characteristics
Histology
Concordance of histologic grade
Histologic grade of index tumor
Concordance of nuclear grade
Nuclear grade of index tumor
Subtype of DCIS a
P53 overexpression

Similar
Different
Same
Different
I or II
III
Same
Different
Low or intermediate
High
Identical to invasive tumor
Mixed
Absent
Present

Same subtype in
tumor foci (n = 60)

Different subtype in
tumor foci (n = 5)

P value

41 (68)
19 (32)
53 (88)
7 (12)
37 (62)
23 (38)
53 (88)
7 (12)
34 (57)
26 (43)
39 (93)
3 (7)
50 (83)
10 (17)

0 (0)
5 (100)
4 (80)
1 (20)
0 (0)
5 (100)
4 (80)
1 (20)
0 (0)
5 (100)
2 (40)
3 (60)
1 (20)
4 (80)

.005

NOTE. Numbers in parentheses indicate column percentages. P values were calculated using Fisher exact test.
a
Subtype of DCIS was evaluated in 47 cases.

.586
.012
.586
.021
.011
.006

54
Six (13%) of 47 cases had mixed molecular subtypes in the
DCIS component, which is similar to the findings of Allred
et al [27]. In that study, diversity of histologic grade,
biomarker phenotype, and intrinsic subtype often existed in
the pure DCIS, and 10% of pure DCIS contained diverse
intrinsic subtypes in nearly all possible combinations, as
defined by immunohistochemistry. Volante et al [28] also
reported, using androgen receptor assays, that some DCIS
and lobular carcinoma in situ arise from different cell clones.
Hypothesizing that pure DCIS gains phenotypic diversity
from different cell clones or from the accumulation of
genetic alterations of a single clone, and then the dominant
clones progress to invasive cancers, it is possible to show
different phenotypes in multifocal/multicentric breast cancer
with a heterogeneous DCIS component.
Some multifocal/multicentric breast cancers arise from
lymphatic or vascular seeding of the index tumor. We found
that 38% of the cases showed lymphovascular invasion,
which is similar to the 44% reported by Middleton et al [10].
If a given multifocal/multicentric breast cancer arises from
intramammary lymphovascular seeding, there is likely to be
a higher risk of further metastases. Reports of a higher
frequency of lymph node involvement and a higher
recurrence rate in multifocal/multicentric breast cancers
than in unifocal breast cancers support the concept that
they can arise by lymphovascular invasion, although the high
incidence of lymph node metastasis in multifocal/multicentric breast cancers is also related to the larger tumor
burden [5,7,29,30].
In summary, we found that ER status was heterogeneous
in 3%, PR in 11%, HER-2 in 6%, and molecular subtype in
8% of multifocal/multicentric IDCs. This result suggests
that multifocal/multicentric IDCs usually have the same
phenotype, but occasionally show discordant phenotype.
Thus, for routine pathology practice, we suggest that
immunohistochemical analysis of multifocal/multicentric
breast cancers can be confined to the index tumor,
especially if they have the same histologic features and
low nuclear/histologic grade.

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