In: Tumor Markers Research Perspectives
Editor: G. A. Sinise, pp. -
ISBN: 978-1-60021-577-3
© 2007 Nova Science Publishers, Inc.
Chapter XIV
Characterization of Breast Cancer
Subtypes by Immunohistochemistry in
a Large Retrospective Study
J. Decock 1, W. Hendrickx 1,2, C. Stefan 1, P. Neven 2, H. Wildiers 1,2,
MR. Christiaens 2, A. Smeets 1,2 and R. Paridaens 1,2
*
1
Laboratory for Experimental Oncology (LEO), Department of General Medical
Oncology, University Hospital Gasthuisberg, Herestraat 49, 3000 Leuven, Belgium
2
Multidisciplinary Breast Center (MBC), University Hospital Gasthuisberg,
Herestraat 49, 3000 Leuven, Belgium
Abstract
Background: DNA microarray studies identified distinct molecular subtypes that
are associated with different clinical outcome, defined as luminal A, luminal B, basal-like
and Her2+ER-. This study aimed to evaluate the immunohistochemistry markers ER, PR
and Her2 as surrogate markers for the previously identified molecular subtypes in a large
retrospective cohort (n=1678) with invasive breast carcinomas with regard to various
demographic, clinical and pathological features.
Methods: All patients were diagnosed with primary breast cancer between 2000 and
2005 at the University Hospital Leuven. None of them received neo-adjuvant therapy, had
bilateral cancer, tumors with direct extension to chest wall or skin, or nipple Paget’s
disease. ER, PR and Her2 expression was determined by immunohistochemistry and cases
with intermediary staining for Her2 were further subjected to two-color fluorescence in
situ hybridization analysis. Clinical and pathological features included age at diagnosis,
menopausal status, maximum tumor size, tumor grade, lymph node status and Nottingham
Prognostic Index (NPI).
*
Corresponding author: Decock J., Laboratory for Experimental Oncology, University Hospital Gasthuisberg KULeuven, O&N1, Room 815, Herestraat 49, 3000 Leuven, Belgium. Phone: +32 (0)16.34.62.93; Fax: +32
(0)16.34.69.01; E-mail: julie.decock@med.kuleuven.be
2
J. Decock, W. Hendrickx, C. Stefan et al.
Results: Of the 1678 patients with invasive breast carcinoma, 80% (n=1342) had
luminal A subtype, 7% (n=119) had luminal B subtype, 9% (n=144) had basal-like type
and 4% (n=73) had Her2+/ER- carcinomas. Within the luminal type, the majority of cases
were PR+, with 87% and 83% for the A and B subtypes, respectively. Luminal A
carcinomas were more often small in size (p=0.037), well/moderately differentiated
(p<0.00001) and associated with a lower NPI value (p<0.00001). Patients with a luminal
B-type tumor presented more frequently with lymph node involvement (p=0.04) and were
significantly younger at diagnosis (p=0.00012) compared to the other patients. Most
basal-like and Her2+/ER- carcinomas were poorly differentiated (92% and 85%
respectively). Furthermore, patients with luminal A or Her2+/ER- carcinomas were more
often postmenopausal than patients with a luminal B or basal-like tumor (p=0.016).
Conclusions: In our large retrospective cohort of 1678 invasive breast cancers, the
luminal A carcinomas were by far best represented in comparison with the other
immunohistochemistry-defined (sub)types, and significantly associated with good
prognostic clinical and pathological parameters. By contrast, although less represented,
the luminal B, the basal-like and the Her2+ER- tumor (sub)types were more frequently
associated with unfavourable prognostic factors such as younger age at diagnosis, lymph
node involvement or poor histological grade.
Keywords: breast cancer, subtypes, clinicopathological parameters, immunohistochemistry.
Introduction
Breast cancer is a heterogeneous disease consisting of several histological subtypes with
different clinical outcome and with patients showing a diverse range of responses to a given
treatment. Although lymph node involvement, histologic grade, tumour size, steroid hormone
receptor expression and Her2 status all have been strongly correlated to prognosis, their
assessment does not alter our inability to accurately predict relapse or response to therapy. A
broad variety of genes and proteins has been analyzed in single-marker prognostic and
predictive studies but most of them were hampered by small size, patient selection criteria,
tumor heterogeneity or treatment diversity and consequentially lacked power to identify all
patients at risk for recurrence and those who would benefit from a new therapy [1; 2]. A large
number of genes are involved in cell growth, death and differentiation and account as such for
much of the phenotypic diversity between tumors. The growing knowledge of these complex
gene signal transduction pathways emphasizes the importance of studying multiple genetic
alterations simultaneously and promotes the emergence of oncogenomics. A major goal in the
field of oncogenomics is to try to answer the clinically important questions about which
tumours will behave aggressively, which tumours will remain dormant, which patients do and
do not require systemic therapy and what type of drugs should be used [3].
Analyses of thousands of genes using microarrays have classified breast carcinomas into
distinct subtypes, associated with different clinical outcomes. Perou and colleagues initially
classified 38 invasive breast carcinomas based on their distinct gene expression profile into
four subtypes: luminal-like, basal-like, Her2+/ER- and normal breast-like [4]. Later the same
research group analyzed the expression profiles of 78 breast carcinomas and categorized them
into five subgroups based on variations in gene expression: luminal A, luminal B, Her2+/ER-,
Breast Cancer Subtypes
3
basal-like and normal breast-like, [5]. Since then, various microarray studies confirmed the
segregation of breast tumors into distinct subtypes with different clinical outcomes in various
ethnic populations [6-11]. Patients with luminal A type tumors are facing a relatively good
prognosis, whereas patients with basal-like tumors experience a much shorter overall and
disease free survival period. Some studies also reported on the clinical and pathological
characteristics of the different subtypes [6; 7; 12-16]. Furthermore, it has been shown that the
various subtypes remarkably differ in their response to treatment, as reviewed by Brenton et
al. [17].
Not surprisingly, hope has risen that this new classification system might improve the
accuracy of prognostic stratification and lead to the development of new tumor-tailored
therapeutic strategies. Although microarrays have the potential to be used as diagnostic and
prognostic tools, they have not yet come into routine clinical practice due to their timeconsuming character and high cost. In this context, the usefullness of immunohistochemical
surrogates for separation of the distinct subtypes should be evaluated, as indicated by the
group of Carey LA [7]. They evaluated the use of immunohistochemistry (IHC) markers that
were previously verified against gene expression profiles to estimate the prevalence of the
intrinsic subtypes in a large population-based epidemiological study of African American and
white women. They identified IHC profiles that best matched the gene expression profiles by
performing both microarray analysis and IHC and validated these IHC surrogates using a
930-case tissue microarray. In this way, IHC-based definitions for the various molecular
subtypes were ER+PR+/-Her2- for luminal A, ER+PR+/-Her2+ for luminal B, ER-PR-Her2+ for
Her2+ subtype and ER-PR-Her2- for basal-like [5; 8; 16].
We aimed to characterize these IHC-defined subgroups on a large retrospective cohort of
1678 invasive breast cancer patients with regard to classic clinical and pathological features.
Materials and Methods
Patients
A total of 1678 patients with primary operable breast cancer were included in this study.
Patient characteristics were extracted from clinical files, tumour characteristics and lymph
node status were retrieved from pathology reports, and all data were collected in our central
breast cancer database. All patients were newly diagnosed at the Multidisciplinary Breast
Center of the University Hospital of Leuven between 2000 and 2005, underwent mastectomy
or local wide excision of their primary breast tumour and axillary lymph node dissection for
staging and treatment. None of them received neo-adjuvant therapy, had bilateral cancer,
tumors with direct extension to chest wall or skin, or nipple Paget’s disease. For all patients
included, data are available on age at diagnosis, menopausal status, tumour size and grade,
lymph node involvement, steroid hormone receptor expression and Her2 status.
4
J. Decock, W. Hendrickx, C. Stefan et al.
Pathological Assessment of Tumour Tissue
Tumour typing and grading were performed on paraffin embedded haematoxylin-eosin
(H&E) slides according to the WHO-classification and the Ellis and Elston grading system
respectively. Lymph nodes were examined with H&E using 3 sections per node. The
Nottingham Prognostic Index was calculated using the equation NPI = 0.2 x tumor size (cm)
+ grade (I–III) + lymph node score.
Immunohistochemical staining for ER, PR and Her2 was performed on 4µm thick serial
paraffin sections. Heat-induced epitope retrieval was carried out in a calibrated water bath
(95-99°C) and antibody complexes were visualised by Envision (Dako, Glostrup, Denmark)
and DAB. A broad variety of antibodies for ER and PR has been used in the period of 2000
to 2005 and semiquantitative evaluation has been performed using the H-score for tumors
resected between January 2000 and august 2003 or the Allred score system for tumors
resected after august 2003. The primary mouse monoclonal antibody CB11 (Novocastra
Laboratories, Newcastle-upon-Tyne, UK) directed against Her2 was applied in a dilution of
1/40 and expression was evaluated with the DAKO scoring system.
Classification of Breast Cancer Subtypes
Based upon ER, PR and Her2 immunohistochemical stainings, breast tumors were
classified into 4 subgroups ER+PR+/-Her2-, ER+PR+/-Her2+, ER-PR-Her2- and ER-PR-Her2+,
which resemble the previously identified luminal A, luminal B, basal-like and Her2+/ERsubgroups. Both the H-scoring and Allred scoring systems take in account the staining
intensity and percentage of cells positively stained. A complete H-score was calculated by
summing the products of the percentage positively stained cells at a given staining intensity
(0–100) and the different staining intensities (0–3). The Allred score was determined by
summing the score representing the proportion of positive tumour cells (0-5) with the staining
intensity (0-3). For both scoring systems, ER and PR expression was considered negative
when staining was completely absent. Using the H score, expression was defined positive
when the final score was 1-300, while for the Allred score expression was considered positive
when the final score was 2-8. The DAKO scoring system for Her2 immunostaining was
applied, taking both the proportion of tumour cells with positive membrane staining and the
staining intensity into account. A score of 0 or 1 was defined negative, while a score of 2 or 3
was considered positive for Her2 overexpression. Cases with a DAKO score of 2 were further
analyzed by 2-color Fluorescence In situ Hybridisation (PathVysion, Vysis, Downers Grove,
IL, USA) in order to distinguish true amplification from polysomie. A mean
Her2/chromosome 17 ratio > 2 was considered amplified for Her2.
Statistical Analyses
Differences between breast cancer subtypes with regard to clinicopathological
characteristics were examined using 1-way ANOVA for age at diagnosis and NPI value and
Chi Square tests for the remaining variables. Statistical analyses were performed using the
software package SPSS version 13, the level of significance being set at p ≤ 0,05.
Breast Cancer Subtypes
5
Results
Study Population
The median age at diagnosis of all patients was 58 (range 26-95). The majority (1177) of
the patients were postmenopausal, while 498 patients were premenopausal. Small and large
tumours were equally distributed, 857 (51%) and 821 (49%) tumours respectively. A total of
231 tumours (14%) were well differentiated, 797 (47%) moderately and 650 (39%) poorly.
No nodal involvement was found in 1069 (64%) patients while 609 (36%) were lymph node
positive. Stratification of tumours into molecular subtype-like groups; based on ER, PR and
Her2 expression; resulted in 1342 (80%) luminal A, 119 (7%) luminal B, 144 (9%) basal-like
and 73 (4%) Her2+/ER- carcinomas. Within the luminal tumor type, the majority of cases
were PR+, with 87% and 83% for the A and B subtypes, respectively (table 1). According to
the American Joint Committee on Cancer staging system, 664 patients had stage I disease,
755 had stage II disease and 259 had stage III disease.
Table 1. Distribution of immunohistochemistry- defined breast cancer subtypes
according to clinical and pathological features (n=1678).
variable
All
n
all
1678
Menopausal status
premenopausal
498
postmenopausal
1177
Histologic
grade
G1+2
1028
G3
650
Tumour size
pT1
857
pT2+3
821
Axillary lymph node status
negative
1069
positive
609
ERPR
ER+PR+
1272
ER+PR189
ER-PR217
Age, mean
58
NPI, mean
4,27
*
luminal A
%(n)
luminal B
%(n)
basal-like
%(n)
p value*
9(144)
Her2+/ER
%(n)
4(73)
80(1342)
7(119)
28
72
39
61
37
63
26
74
0,015
72
28
31
69
8
92
15
85
<0,00001
53
47
45
55
44
56
42
58
0,037
65
35
52
48
65
35
60
40
0,044
87
13
0
59
4,1
83
17
0
54
4,85
0
0
100
56
4,97
0
0
100
57
5,02
<0,00001
0,00012
<0,00001
Associations between immunohistochemistry-defined subtypes and the continuous variables age
and NPI were analyzed using one-way ANOVA, while associations with the remaining
characteristics were analyzed using the Pearson Chi Square test.
6
J. Decock, W. Hendrickx, C. Stefan et al.
Steroid Hormone Receptor Status of Tumor Subtypes
Based on ER, PR and Her2 expression of the tumor, breast tumors can be classified into
immunohistochemistry-defined subtypes. Estrogen receptor negative tumors can be
subdivided into basal-like (ER-PR-Her2-) and Her2+/ER- (ER-PR-Her2+) tumors, while ER+
tumors can further be classified as luminal A (ER+PR+/-Her2-) or luminal B (ER+PR+/-Her2+).
Within the luminal type, we determined the proportion of PR+ cases and found 87% and 83%
of luminal A and luminal B tumors to be PR+ (table 1).
Characterization of Tumor Subtypes
Characteristics of the IHC-defined breast cancer subtypes are presented in table 1. Breast
cancer subtypes differed significantly by age (p=0.0001), menopausal status (p=0.015),
histologic grade (p< 0.00001), tumor size (p=0.037), axillary lymph node status (p= 0.044)
and NPI (p< 0.00001). As the NPI value is based on tumor size, histologic grade and lymph
node involvement, this result reflects the significant difference found among the various
subtypes for each of these parameters.
The proportion of smaller tumors (< 21 mm) was not dramatically different among the
various groups, with the exception again of the luminal A group in which the tumors tended
to be smaller. Luminal carcinomas and in particular luminal A-type tumors were more likely
well/moderately differentiated, whereas the majority of basal-like (92%) and Her2+/ER(85%) carcinomas were poorly differentiated. Patients with a luminal B type tumor had the
highest prevalence of lymph node involvement and were significantly younger than the other
patients. All 4 breast tumor subtypes were associated with a NPI value indicative for a
moderately good prognosis (3.4<NPI<5.4) with the lowest value observed in the luminal Atype tumors. This result for luminal A tumors is not surprisingly since the tumor size, grade
and lymph node involvement in this group of tumors all indicated a good prognosis. The
number of premenopausal patients was higher in the groups with luminal B and basal-like
tumors compared to those with luminal A or Her2+/ER- tumors.
Moreover, each of these findings was also observed in the subgroup of patients with early
stage disease (stage I and II) at an even higher significance, with exception of the lymph node
status (table 2).
Breast Cancer Subtypes
7
Table 2. Characteristics of immunohistochemistry-defined subtypes in breast cancer
patients with early stage disease (n=1419).
variable
all
n
1419
all
Menopausal status
premenopausal
399
postmenopausal
1020
Histologic grade
G1+2
891
G3
528
Axillary lymph node status
negative
1069
positive
350
ERPR
ER+PR+
1078
ER+PR161
ER-PR180
Age, mean
59
NPI, mean
3,91
*
luminal A
%(n)
81(1145)
luminal B
%(n)
7(94)
basal-like
%(n)
7(123)
Her2+/ER%(n)
4(57)
p value*
26
74
40
60
37
63
25
75
0,002
74
26
35
65
6
94
16
84
<0,00001
76
24
66
34
76
24
77
23
0,19
87
13
0
59
3,75
82
18
0
54
4,4
0
0
100
56
4,69
0
0
100
58
4,57
<0,00001
<0,00001
<0,00001
Associations between immunohistochemistry-defined subtypes and the continuous variables age
and NPI were analyzed using one-way ANOVA, while associations with the remaining
characteristics were analyzed using the Pearson Chi Square test.
Conclusion
We examined the presence and characteristics of various immunohistochemistry-defined
subtypes in a large retrospective cohort of invasive breast cancer patients by means of routine
immunohistochemical staining for ER, PR and Her2. We were able to confirm the existence
of four tumor subgroups, resembling the microarray identified subtypes luminal A, luminal B,
basal-like and Her2+/ER-. On basis of their clinical and pathological profile, patients with
luminal A tumors seem to have a good prognosis which was reflected in menopausal status,
small tumor size, high proportion of well/moderately differentiated tumors and consequently a
lower NPI value. In contrast, patients with luminal B tumors were more likely associated with
poor prognosis as they were significantly younger than the others and were often associated
with lymph node involvement. However, any conclusion regarding menopausal status rather
than age in association with breast cancer phenotypes may have been biased by use of
contraceptives or hormone replacement therapy. The differences in clinical prognostic
features for both luminal subtypes can also in part be explained by microarray analyses which
showed that luminal A type tumors have, in general, higher expression of estrogen responsive
genes and lower expression of proliferative genes than luminal B [5; 8].
Just as others, we were not able to clearly characterize the ER- tumor subtypes, basal-like
and Her2+/ER-, except for the observation that they were more likely poorly differentiated [5;
7; 17]. The poor prognosis of the Her2+/ER- and basal-like tumor subtypes is most probably
8
J. Decock, W. Hendrickx, C. Stefan et al.
due to the fewer treatment options available for ER- and PR- tumors. Moreover, although
treatment with the anti-Her2 monoclonal antibody trastuzumab in combination with
chemotherapy significantly improves disease-free-survival among women with advanced
breast cancer and remarkable reduces the number of patients with relapse, not all Her2+
tumors respond to trastuzumab [18].
To our knowledge, this is by far the largest retrospective cohort of invasive breast cancer
patients with clinicopathological characterization of the various tumor subtypes. Although the
molecular breast tumor subtypes were initially identified by microarray gene expression
studies, we support Carey LA and colleagues in their hypothesis that IHC assessment of ER,
PR and Her2 is a good alternative approach for analysis of the subtypes in a large
retrospective cohort. Moreover, our findings are in line with the results from Calza et al. who
recently reported the largest microarray study on a population-based cohort of 412 breast
cancer patients [6]. They observed that luminal A carcinomas were mostly found in
postmenopausal women, tended to be small and were more likely good/moderately
differentiated. Furthermore, they found that in comparison with luminal A tumors, the luminal
B group had a higher proportion of poorly differentiated and lymph node positive tumors. In
their cohort, basal-like tumors were most often found in younger, premenopausal patients
with poorly differentiated tumors, while Her2+/ER- tumors were more frequently observed in
elderly women with large and poorly differentiated tumors. The strength of our study is that
the cohort consisted of as well 1419 (85 %) patients with early stage (stage I, II) disease as
259 (15%) patients with advanced disease (stage III). Interestingly, analysis of early stage
disease revealed the presence of the same four distinct tumor subtypes which were similarly
characterized as those in the overall study population. Other groups analyzed the gene
expression in DCIS for comparison with normal and invasive carcinomas in various ethnic
populations and found that DCIS tumors could be similarly divided into distinct subtypes with
different clinical outcomes [19-21]. All these studies imply that the molecular signatures
defining the subtype of a tumor and its clinical characteristics may already be set in early
stage disease and even at the pre-invasive stage of carcinogenesis.
We would like to emphasize the importance of the methodology used for steroid hormone
and Her2 assessment; immunohistochemical analyses have their limitations. Also, the current
reproducibility and clinical relevance of microarray generated data have recently been
criticized by Dupuy and Simon [22]. Both methodologies are used to measure the gene or
protein expression of ER, PR and Her2 and not necessarily reflect the activity of the receptor
and its downstream pathways. Moreover, immunohistochemical staining of Her2 does not
allow us to distinguish between true gene amplification and polysomy and hence FISH
analysis is required for cases with intermediary Her2 staining. In our institute, dual-color
FISH is therefore performed as routine clinical practice for Her2 overexpressing tumors with
an IHC score of 2. Expression analysis of downstream signalling pathways might enlighten
the knowledge on breast cancer behaviour and improve prognostic accuracy.
In conclusion, using routine immunohistochemistry we were able to confirm the presence
of four subgroups, resembling the microarray identified molecular subtypes, in a large
population-based cohort of breast cancer patients with early or late stage disease.
Furthermore, our study supports the hypothesis that these immunohistochemistry-defined
tumor subtypes are distinct biological entities with distinct clinical characteristics.
Breast Cancer Subtypes
9
Acknowledgements
We gratefully thank Prof. M. Drijkoningen for the pathological assessment of all tumor
specimens. Further, we acknowledge all collaborators of the Multidisciplinary Breast Center,
General Medical Oncology and Gynecology for their help with data input.
Grant support: This work is funded by the “Vlaamse Liga tegen Kanker” and the EU
Framework Programme 6 (LSHC-CT-2003-503297, Cancerdegradome).
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