Jumppanen M, Gruvberger-Saal S, Kauraniemi P, Tanner M, Bendahl PO, Lundin M, Krogh M, Kataja P, Borg A, Ferno M, Isola JBasal-like phenotype is not associated with patient survival in estrogen-receptor-negative breast cancers. Breast Cancer Res 9: R16

Abstract

Basal-phenotype or basal-like breast cancers are characterized by basal epithelium cytokeratin (CK5/14/17) expression, negative estrogen receptor (ER) status and distinct gene expression signature. We studied the clinical and biological features of the basal-phenotype tumors determined by immunohistochemistry (IHC) and cDNA microarrays especially within the ER-negative subgroup.
IHC was used to evaluate the CK5/14 status of 445 stage II breast cancers. The gene expression signature of the CK5/14 immunopositive tumors was investigated within a subset (100) of the breast tumors (including 50 ER-negative tumors) with a cDNA microarray. Survival for basal-phenotype tumors as determined by CK5/14 IHC and gene expression signature was assessed.
From the 375 analyzable tumor specimens, 48 (13%) were immunohistochemically positive for CK5/14. We found adverse distant disease-free survival for the CK5/14-positive tumors during the first years (3 years hazard ratio (HR) 2.23, 95% confidence interval (CI) 1.17 to 4.24, p = 0.01; 5 years HR 1.80, 95% CI 1.02 to 3.15, p = 0.04) but the significance was lost at the end of the follow-up period (10 years HR 1.43, 95% CI 0.84 to 2.43, p = 0.19). Gene expression profiles of immunohistochemically determined CK5/14-positive tumors within the ER-negative tumor group implicated 1,713 differently expressed genes (p < 0.05). Hierarchical clustering analysis with the top 500 of these genes formed one basal-like and a non-basal-like cluster also within the ER-negative tumor entity. A highly concordant classification could be constructed with a published gene set (Sorlie's intrinsic gene set, concordance 90%). Both gene sets identified a basal-like cluster that included most of the CK5/14-positive tumors, but also immunohistochemically CK5/14-negative tumors. Within the ER-negative tumor entity there was no survival difference between the non-basal and basal-like tumors as identified by immunohistochemical or gene-expression-based classification.
Basal cytokeratin-positive tumors have a biologically distinct gene expression signature from other ER-negative tumors. Even if basal cytokeratin expression predicts early relapse among non-selected tumors, the clinical outcome of basal tumors is similar to non-basal ER-negative tumors. Immunohistochemically basal cytokeratin-positive tumors almost always belong to the basal-like gene expression profile, but this cluster also includes few basal cytokeratin-negative tumors.

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Vol 9 No 1
Research article
Basal-like phenotype is not associated with patient survival in
estrogen-receptor-negative breast cancers
Mervi Jumppanen1,2, Sofia Gruvberger-Saal3, Päivikki Kauraniemi2, Minna Tanner2,4, Pär-
Ola Bendahl3, Mikael Lundin5, Morten Krogh6, Pasi Kataja2, Åke Borg3, Mårten Fernö3 and
Jorma Isola2
1Department of Pathology, Seinäjoki Central Hospital, Hanneksenrinne 7, FIN-60220 Seinäjoki, Finland
2Institute of Medical Technology, University and University Hospital of Tampere, Biokatu 6, FIN-33520 Tampere, Finland
3Department of Oncology, Clinical Sciences, University of Lund, Klinikgatan 7, SE-221 85 Lund, Sweden
4Department of Oncology, Tampere University Hospital, Teiskontie 35, FIN-33520 Tampere, Finland
5Biomedical Informatics group, Department of Oncology, University of Helsinki, Haartmanninkatu 8, FIN-00290 Helsinki, Finland
6Department of Theoretical Physics, Lund University, Sölvegatan 14A, SE-221 85 Lund, Sweden
Corresponding author: Mervi Jumppanen, mervi.jumppanen@epshp.fi
Received: 17 Aug 2006 Revisions requested: 31 Oct 2006 Revisions received: 12 Dec 2006 Accepted: 31 Jan 2007 Published: 31 Jan 2007
Breast Cancer Research 2007, 9:R16 (doi:10.1186/bcr1649)
This article is online at: http://breast-cancer-research.com/content/9/1/R16
© 2007 Jumppanen et al.; licensee BioMed Central Ltd.
This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Introduction Basal-phenotype or basal-like breast cancers are
characterized by basal epithelium cytokeratin (CK5/14/17)
expression, negative estrogen receptor (ER) status and distinct
gene expression signature. We studied the clinical and
biological features of the basal-phenotype tumors determined
by immunohistochemistry (IHC) and cDNA microarrays
especially within the ER-negative subgroup.
Methods IHC was used to evaluate the CK5/14 status of 445
stage II breast cancers. The gene expression signature of the
CK5/14 immunopositive tumors was investigated within a
subset (100) of the breast tumors (including 50 ER-negative
tumors) with a cDNA microarray. Survival for basal-phenotype
tumors as determined by CK5/14 IHC and gene expression
signature was assessed.
Results From the 375 analyzable tumor specimens, 48 (13%)
were immunohistochemically positive for CK5/14. We found
adverse distant disease-free survival for the CK5/14-positive
tumors during the first years (3 years hazard ratio (HR) 2.23,
95% confidence interval (CI) 1.17 to 4.24, p = 0.01; 5 years HR
1.80, 95% CI 1.02 to 3.15, p = 0.04) but the significance was
lost at the end of the follow-up period (10 years HR 1.43, 95%
CI 0.84 to 2.43, p = 0.19). Gene expression profiles of
immunohistochemically determined CK5/14-positive tumors
within the ER-negative tumor group implicated 1,713 differently
expressed genes (p < 0.05). Hierarchical clustering analysis
with the top 500 of these genes formed one basal-like and a
non-basal-like cluster also within the ER-negative tumor entity. A
highly concordant classification could be constructed with a
published gene set (Sorlie's intrinsic gene set, concordance
90%). Both gene sets identified a basal-like cluster that
included most of the CK5/14-positive tumors, but also
immunohistochemically CK5/14-negative tumors. Within the
ER-negative tumor entity there was no survival difference
between the non-basal and basal-like tumors as identified by
immunohistochemical or gene-expression-based classification.
Conclusion Basal cytokeratin-positive tumors have a
biologically distinct gene expression signature from other ER-
negative tumors. Even if basal cytokeratin expression predicts
early relapse among non-selected tumors, the clinical outcome
of basal tumors is similar to non-basal ER-negative tumors.
Immunohistochemically basal cytokeratin-positive tumors almost
always belong to the basal-like gene expression profile, but this
cluster also includes few basal cytokeratin-negative tumors.
Introduction
cDNA microarray studies have shown that the most powerful
denominator in determining the gene expression profiles and
prognostic groups of breast cancer is estrogen receptor (ER)
and ER-related genes [1-5]. Breast cancers have been sepa-
rated by gene expression profiles into luminal, basal-like,
CI = confidence interval; CISH = chromogenic in situ hybridization; EASE = Expression Analysis Systematic Explorer; EGFR = epidermal growth
factor receptor; ER = estrogen receptor; GO = gene ontology; HR = hazard ratio; IHC = immunohistochemistry; TMA = tissue microarray.

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ERBB2, and normal breast-like subgroups [6-9]. Basal-like
tumors express many of the genes characteristic of breast
basal epithelial cells [6] and the most typical feature of basal-
like breast cancers is the lack of expression of ER and genes
usually co-expressed with ER [6-9].
In addition to the gene expression microarray studies, basal-
phenotype breast tumors have long been identified by using
basal cytokeratin immunohistochemistry (IHC) [10-20]. Basal
cytokeratin (CK5/14/17)-positive tumors represent about
10% of sporadic breast carcinomas and are almost exclusively
ER-negative, poorly differentiated, and associated with epider-
mal growth factor receptor (EGFR), p53, vimentin, and c-kit
immunopositivity and Bcl-2 negativity [11,12,14-16,19-21].
Even though gene expression studies separate the basal-like
tumors from the ERBB2 tumor subgroup [6-9], there are some
immunohistochemically basal cytokeratin-expressing tumors
that show HER-2 oncogene amplification [12,17,22]. The
relationship between immunohistochemical and microarray-
based classification of basal-phenotype breast cancer has not
been established.
Apart from hypothesis-generating scientific research, a breast
tumor classification should correlate with the clinical outcome
of patients or predict efficacy to therapy. Negative ER status,
which is the most prominent feature of basal-phenotype
tumors, is a well-established prognostic and predictive factor
in breast cancer. Microarray studies have shown that basal-like
tumors have poor prognosis when compared with ER-positive
luminal tumor groups but not when compared with a ERBB2
tumor cluster [7,8]. Immunohistochemical studies with basal
cytokeratin IHC for the basal breast cancer phenotype classi-
fication have almost exclusively addressed the fact that basal-
phenotype tumors have poor prognosis, but they have also
made the comparison in cohorts not selected by matching ER
status (ER-negative) [10,11,16,17,20,23-25]. In this study we
defined the gene expression profile of basal cytokeratin immu-
nopositive tumors and studied the clinical outcome especially
within the ER-negative tumor entity.
Materials and methods
Tumor samples
The tumor cohort comprised 445 primary stage II breast can-
cers collected from the South Sweden Health Care Region
between 1985 and 1994 with approval from the Lund Univer-
sity Hospital ethics committee; the cohort was described pre-
viously in more detail by Chebil and colleagues [26]. In the
present study, patients treated with 20 mg of tamoxifen daily
for 2 years with complete follow-up data and uniform immuno-
histochemical method for hormone receptor analysis were
included. Radical mastectomy or breast-conserving surgery
was used with axillary lymph node dissection. Radiotherapy
was introduced for all patients treated with breast-conserving
surgery and for patients with lymph-node-positive disease. The
patients were not treated with adjuvant chemotherapy. The
median follow-up time for distant disease-free survival was 6
years.
Immunohistochemistry
The formalin-fixed paraffin-embedded sample material was
provided as eight tissue microarrays (TMAs) containing three
core samples (diameter 0.6 mm) for each primary tumor.
Immunohistochemical staining with CK5/CK14/p63 antibody
cocktail (XM26, dilution 1:400, Novocastra, Newcastle upon
Tyne, UK; LL002, dilution 1:400, Novocastra; 4A4+Y4A3,
dilution 1:1,500, Neomarkers, Fremont, CA, USA, respec-
tively) and with p53 antibody (DO-7, dilution 1:500, Novocas-
tra) was performed as described previously [12,22]. Hormone
receptors (ER and progesterone receptor) were conducted
earlier by IHC from the original tissue blocks as described by
Chebil and colleagues [26].
Analysis of the HER-2 oncogene amplification was conducted
by using a chromogenic in situ hybridization (CISH) method as
described previously [27]. The histological type of the tumors
was determined in accordance with the WHO classification as
described by Chebil and colleagues [26].
Sample scoring
Immunohistochemically stained TMA samples for CK5/CK14/
p63 and p53 as well as HER-2 CISH stainings were scanned
with a virtual microscopy technique as described previously
[28]. Immunostaining for CK5/CK14/p63 was considered
CK5/14-positive if at least 20% of the tumor cells showed
cytoplasmic staining and positive for p63 when the staining
was nuclear. p53 was regarded as positive when at least 20%
of the tumor cells were stained. HER-2 oncogene was consid-
ered amplified if six or more gene copies were found per cell
in at least 10% of the tumor cells.
Statistical analysis
Fisher's exact test and the χ2 test were used to test the signif-
icance of the cross-tabulated data (using Stata 9.2 (Stata Cor-
poration, College Station, TX, USA) and MedCalc (MedCalc
Software, Mariakerke, Belgium) statistical software packages).
Survival analyses were calculated with Kaplan-Maier life table
curves, a log-rank test and a univariate Cox model. Distant dis-
ease-free survival was calculated from the primary diagnosis to
the date of an event (distant recurrence or death) or, for event-
free patients, to the date of the most recent follow-up. All
reported p values are two-sided.
Gene expression microarrays
cDNA microrrays were manufactured in the SWEGENE
Microarray Facility, Department of Oncology, Lund University.
The gene set consisted of 24,301 sequence-verified IMAGE
clones (Research Genetics, Huntsville, AL, USA) and 1,296
internally generated clones, together representing about
16,000 Unigene clusters (build 180) and about 1,200 unclus-
tered expressed sequence tags. The clones were amplified by

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polymerase chain reaction with vector-specific primers essen-
tially as described previously [29].
A selected subset (n = 100, of which 50 were ER-negative)
from the total cohort was analyzed with microarrays. Nineteen
of these tumors showed positive CK5/14 staining and the rest
were negative. Only one of the CK5/14-positive tumors was
ER-positive. Total RNA was extracted from grossly dissected
frozen tissue samples (about 100 mg) by the subsequent use
of Trizol (Invitrogen, Carlsbad, CA, USA) and the RNeasy kit
(Qiagen, Hilden, Germany). For each hybridization, 15 μg of
Universal Human Reference RNA (Stratagene, La Jolla, CA,
USA) was used to synthesize reference Cy5-labeled targets
and 25 μg of sample total RNA for Cy3-labeled targets.
Anchored oligo(dT) primers, the CyScribe indirect amino-allyl
cDNA synthesis and labeling protocol and GFX purification
columns (Amersham Biosciences, Little Chalfont, Bucks., UK)
were used. Together with blocking agents (12 μg of poly-(dA),
6 μg of yeast tRNA, and 20 μg of Cot-1 DNA), targets were
hybridized to the microarrays for 18 hours under a glass cov-
erslip with the use of humidified Corning hybridization cham-
bers at 42°C and the Pronto Universal Hybridization System
(Corning Inc., Corning, NY, USA). Slides were scanned at 10
μm resolution in an Agilent DNA Microarray Scanner (Agilent
Technologies, Palo Alto, CA, USA) and the images were ana-
lyzed with GenePix Pro software (Axon Instruments, Union
City, CA, USA).
Microarray data analysis
The data were analyzed with BASE (BioArray Software Envi-
ronment) software [30]. In brief, background-corrected inten-
sities for sample and reference channels were calculated by
subtracting the median local background signal from the
median foreground signal for each spot. Filters were applied to
remove all spots flagged during image analysis. Data within
individual arrays were then normalized by using an implemen-
tation of the 'lowess' (locally weighted linear regression) algo-
rithm [31]. Poorly measured/expressed spots with a signal-to-
noise ratio of 3 or less in either the Cy3 or Cy5 channel were
removed, and genes with missing data in more than 20% of all
arrays or genes with a variation across arrays of not more than
0.45 standard deviations of the log2(ratio) were filtered, leav-
ing 10,479 informative genes. The expression ratios for each
gene were then median-centered across all tumors.
To generate a gene list for the basal-phenotype tumors, corre-
lation scores were calculated between gene expression
(log2(ratio)) for all reporters and the CK5/14 immunopositive
tumors [32]. To evaluate the significance of the expression sig-
natures between the two annotation classes (CK5/14-positive
and CK5/14-negative), 1,000 permutations were run in which
the samples were randomly given an annotation label, and the
p value for a score was calculated as the average number of
reporters exceeding the score in the permutation test, divided
by the total number of reporters in the gene list. The false dis-
covery rate – that is, the estimated number of genes in a given
set of scored genes that could receive an equal or better score
by chance – was calculated by random permutations and used
as an indicator of the robustness of the gene expression pro-
file. A false discovery rate of 0% indicates no false positives; a
false discovery rate of 100% indicates a completely random
signal. Gene expression profiles were analyzed with hierarchi-
cal clustering with centered Pearson correlation and average
linkage clustering [33].
The ranked gene list was subjected to gene ontology annota-
tion analysis with EASE (Expression Analysis Systematic
Explorer) [34], in which only biological process ontology cate-
gories were included and the enrichment of categories in the
gene list was evaluated by comparison with the total list of
genes used for the microarray analysis. An EASE score of p ≤
0.05 was considered to be significant. The UniGene clusters
representing the top 200 genes were annotated with subcel-
lular location by cross-reference to two published microarray
datasets [33,35] and to Swiss-Prot. The Swiss-Prot Subcellu-
lar Locations annotations were downloaded from the
DRAGON database [36]. A gene was considered to be mem-
brane associated or secreted if the Swiss-Prot annotation con-
tained one of the words 'membrane', 'vesicle', or 'secreted', or
if the membrane:cytosolic ratio in the polysome fraction study
exceeded 2 or 1.08 in the studies by Diehn and colleagues
[35] or Stitziel and colleagues [37], respectively. Primary
expression data are available from the NCBI Gene Expression
Omnibus database (accession ID GSE6768) [38].
Results
Immunohistochemical detection of basal-phenotype
tumors
Immunohistochemical analysis was performed on TMAs con-
taining 445 tumors, of which 375 (84%) were analyzable for
CK5/CK14/p63 antibody cocktail. There were 48 (13%)
CK5/14-positive and 13 (3.5%) p63-positive tumors.
Although CK5/14 and p63 are co-expressed in normal cells of
breast ducts, there was no association in malignant epithelial
cells (p = 0.22). The CK5/14 immunopositivity was signifi-
cantly correlated with negative ER status (p < 0.0001, data
not shown). There were 13 ER-positive basal cytokeratin-
expressing tumors. Association with negative progesterone
receptor status (p < 0.0001) with negative lymph node status
(p = 0.0005) and with p53 immunopositivity (p = 0.003) was
also seen but there was no association with HER-2 oncogene
amplification (p = 0.80, data not shown). Among the 95 ER-
negative tumors, 35 (37%) showed positive staining for CK5/
14 (Table 1). When CK5/14 positivity was correlated with clin-
icopathological characteristics within the ER-negative tumor
subgroup, associations with negative lymph node status and
positive p53 status were not seen (p = 0.14 and p = 0.65,
respectively), but significant association between CK5/14
immunopositivity and negative HER-2 status emerged (p =
0.01, Table 1). Most of the basal cytokeratin-positive tumors

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were of the ductal histotype (80%) and the rest were of the
medullary or atypical medullary histotypes (20%; Table 1).
Over half (7/12) of the medullary histotype tumors (medullary
or atypical medullary) were in fact CK5/14-positive.
Gene expression profile of basal-phenotype tumors
A clear difference was seen in gene expression profiles
between the basal cytokeratin (CK5/14) immunopositive and
negative subgroups in the whole data set (false discovery rate
0.03% for the 100 genes, and 0.3% for the top 500 with the
use of the Golub algorithm) including both ER-positive and
ER-negative tumors. However, because the basal phenotype
determined by IHC was strongly correlated with negative ER
status (only one of the 50 ER-positive tumors stained positive
for CK5/14), and because ER status has been shown to have
a strong influence on the gene expression signature of breast
tumors [2,4,6], we performed an analysis in the subset of ER-
negative tumors (n = 50) separately. In this subset CK5/14-
positive and CK5/14-negative tumors were also associated
with two distinct gene expression signatures (false discovery
rate 6.7% for the top 100 genes and 16.1% for the top 500
genes). Hierarchical clustering analysis of the ER-negative
tumors using the top 500 basal discriminatory genes gener-
ated within the ER-negative tumor group identified two sepa-
rate clusters (Figure 1; see Additional file 1 for the heat map):
one cluster containing a large number of CK5/14-positive
tumors (17/24) in addition to seven CK5/14-negative tumors,
and another in which all except one of the tumors (25/26)
were immunohistochemically CK5/14-negative and were fre-
quently amplified for the HER-2 oncogene (18/26). Although
the signal for the basal phenotype among ER-negative tumors
was weaker than in the whole data set, in which the classifica-
tion may have been highly influenced by the strong ER-related
signal, it was statistically highly significant (1,713 genes were
identified with p < 0.05; see Additional file 2 for the top 200
genes).
We next explored how the so-called 'intrinsic' gene set gener-
ated by Perou and colleagues [6-8] would perform in our data
set. Mapping of their intrinsic gene list [8] to our data with the
use of Unigene Cluster ID as an identifier produced a list of
522 clones. These clones were used to cluster the whole data
set, which gave expected results separating basal/ER-, lumi-
nal/ER+ and ERBB2+/ER- tumor groups from each other sim-
Table 1
Clinicopathological characteristics of estrogen-receptor-negative breast tumors according to basal cytokeratin (CK5/14) status
Clinicopathological parameter CK5/14 negative (percentage) CK5/14 positive (percentage) p
All estrogen-receptor-negative tumors 60 (63) 35 (37)
Axillary lymph node status
Negative 23 (38) 19 (54) 0.14
Positive 37 (62) 16 (46)
HER-2 status
Non-amplified 35 (58) 30 (86) 0.01
Amplified 18 (30) 3 (9)
Data missing 7 (12) 2 (6)
p63
Negative 57 (95) 33 (94) 1.00
Positive 3 (5) 2 (6)
p53
Negative 26 (43) 13 (37) 0.65
Positive 26 (43) 17 (49)
Data missing 8 (13) 5 (14)
Histological type
Invasive ductal or mixed type 49 (82) 28 (80) 0.10
Invasive lobular 5 (8) 0 (0)
Medullary or atypical medullary 5 (8) 7 (20)
Other types 1 (2) 0 (0)
The p values were calculated, excluding the 'data missing' values, with Fisher's exact test except for the p value of histological type, which was
calculated with the χ2 test.

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ilarly to the original study (data not shown) [6,8]. Hierarchical
clustering of the ER-negative tumor group separately, with the
use of the intrinsic gene set, generated a dendrogram with two
major subgroups very similar to the hierarchical clustering
analysis with our top 500 ranked basal genes (concordance
90%, p = 0.0001; Figure 2). The basal-like cluster included
most of the CK5/14-positive tumors and nine additional CK5/
14-negative tumors. The tumors in the non-basal subgroup
showed frequent HER-2 amplification (17/27) and predomi-
nantly a CK5/14-negative immunophenotype (23/27; Figure
2; see Additional file 3 for the heat map). The basal phenotype
classification by Sorlie's intrinsic gene set correlated strongly
with basal cytokeratin IHC (concordance 76%, p = 0.0011).
Interestingly, seven of the nine misclassified CK5/14-negative
tumors by Sorlie's intrinsic gene set were also found to belong
to the basal-like cluster when our top 500 CK5/14-associated
genes were used in hierarchical clustering analysis.
The gene list generated for the basal cytokeratin immunopos-
itive tumors within the ER-negative tumor entity (Additional file
2) included genes associated with ER status such as TTF1
(rank 13) and XBP1 (rank 16) and other genes previously
associated with the basal-like tumor subtype such as CRYAB
(rank 26), TRIM29 (rank 51), ERBB2 (rank 55), ANXA8 (rank
134), and EGFR (rank 193) [6-9]. Twelve of the genes with a
high expression in basal-like tumors (within the top 200 genes)
were annotated as having a membrane-bound cellular localiza-
tion, but not to the mitochondria or the Golgi apparatus (Addi-
tional file 2).
Distant disease-free survival of basal-phenotype tumors
Association of the basal status with patient prognosis was
evaluated first in the immunohistochemically defined basal
(CK5/14-positive) and non-basal (CK5/14-negative) tumor
subgroups. In the whole tumor material, the distant disease-
free survival was significantly shorter for the CK5/14-positive
tumors during the first years of follow-up (3 years hazard ratio
(HR) 2.23, 95% confidence interval (CI) 1.17 to 4.24, p =
0.01 and 5 years HR 1.80, 95% CI 1.02 to 3.15, p = 0.04),
but this difference was lost at the end of the follow-up period
(10 years HR 1.43, 95% CI 0.84 to 2.43, p = 0.19; Figure 3).
Next we studied clinical outcome within the ER-negative entity.
The survival rates of immunohistochemically CK5/14-positive
and CK5/14-negative tumor groups were identical, as
Figure 1
Hierarchical clustering of 50 ER-negative tumors based on the top 500 basal genesHierarchical clustering of 50 ER-negative tumors based on the top 500 basal genes. The gene set was generated for the CK5/14-positive basal
phenotype tumors. Yellow indicates the basal-like cluster and black the non-basal-like cluster. The boxes beneath indicate the immunohistochemi-
cally CK5/14-positive tumors and the HER-2 oncogene-amplified tumors (solid box, positive; open box, negative, crossed box, data missing).
Figure 2
Hierarchical clustering of 50 ER-negative tumors based on the intrinsic gene set [7]Hierarchical clustering of 50 ER-negative tumors based on the intrinsic gene set [7]. Yellow indicates the basal-like cluster and black the non-basal-
like cluster. The black boxes beneath indicate the basal-like cluster by the top 500 basal genes, immunohistochemically CK5/14-positive tumors,
and HER-2 amplified tumors (solid box, positive; open box, negative, crossed box, data missing).

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demonstrated by the superimposed Kaplan-Meier curves and
log-rank test (p = 0.93; Figure 4a). The same result was
obtained when the basal-like classification was based on gene
expression microarrays (p = 0.42 and p = 0.55 for classifica-
tions based on our gene list and Sorlie's gene list (Figure
4b,c), respectively).
Functional analysis of genes aberrantly expressed in
basal-phenotype tumors
We next performed a gene ontology (GO) annotation analysis
of the top 1,000 genes on our basal gene list (within ER-neg-
ative tumors) and found that 823 genes were associated with
a functional gene annotation category. Of these genes, 383
were upregulated in the CK5/14-positive tumors and 440
were downregulated (Additional file 4). Genes upregulated in
basal-like tumors (with an EASE score of 0.05 or less)
belonged to the annotation categories epidermal differentia-
tion (GO:0008544) and ectoderm development
(GO:0007398), protein biosynthesis (GO:0006412), nuclear
division (GO:0000280), development (GO:0007275), bio-
synthesis (GO:0009058), histogenesis (GO:0009888), mac-
romolecule biosynthesis (GO:0009059), and M phase
(GO:0000279). Basal cytokeratins 14 and 17 were present in
the gene category of epidermal differentiation and ectoderm
development, which was the most significantly upregulated
biological process in basal-phenotype tumors. Genes down-
regulated in basal-phenotype tumors were characterized as
having functions in cell-surface receptor-linked signal trans-
duction (GO:0007166), enzyme-linked receptor protein sign-
aling pathway (GO:0007167), transmembrane receptor
protein tyrosine kinase signaling pathway (GO:0007169), and
regulation of G-protein-coupled receptor protein signaling
pathway (GO:0008277).
Discussion
Basal-like breast cancer has been associated with poor prog-
nosis in several immunohistochemical [10,11,15-18,20,22-
25] and gene expression microarray-based studies [7-9]. Nev-
ertheless, there are conflicting results between studies about
the independent prognostic significance of the basal pheno-
type [11,15,18,20]. Adjuvant chemotherapy could be recog-
nized as one possible confounding factor, because it has been
postulated that basal-like and non-basal tumors would
respond differently to chemotherapy [39]. Our results showed
that when using IHC to identify basal-like tumors, a survival dif-
ference was seen in the entire patient population during the
first years of the follow-up. This suggests that basal cytokeratin
expression predicts early relapse when compared with non-
basal tumors, including both ER-positive and ER-negative
Figure 3
Distant disease-free survival of immunohistochemically CK5/14-nega-tive and CK5/14-positive tumors in the whole data setDistant disease-free survival of immunohistochemically CK5/14-nega-
tive and CK5/14-positive tumors in the whole data set. The basal cytok-
eratin-positive tumors show significantly shorter survival during the first
years of the follow-up, but this difference is lost with time.
Figure 4
Distant disease-free survival of basal-like and non-basal-like tumors within the ER-negative tumor entityDistant disease-free survival of basal-like and non-basal-like tumors
within the ER-negative tumor entity. The basal phenotype was defined
by using immunohistochemistry (a), cDNA microarray and the top 500
gene set for the basal cytokeratin-immunopositive tumors (b) or cDNA
microarray and the intrinsic gene set of Sorlie and colleagues [7] (c).
There is no difference in survival between basal-like and non-basal-like
tumors within the ER-negative tumor subgroup.

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breast cancers. This is in agreement with previous results
[11,15-18,20,22-25]. Furthermore, our tumor series repre-
sents early-stage disease not treated with chemotherapy. It
therefore presents a more coherent picture of the natural biol-
ogy of breast cancer than when studying chemotherapy-
treated patients. It must still be noted that in this study all the
patients were treated with tamoxifen for 2 years, which most
probably affected the natural history of the ER-positive tumors.
Even though we saw a survival difference between basal and
non-basal tumors when studying the whole population, this
was not true within the ER-negative tumor subgroup. This
therefore suggests that basal cytokeratin expression is not an
independent prognostic factor. Our results support the find-
ings of Potemski and colleagues [18] and Malzahn and col-
leagues [15], who did not find any difference between basal
and non-basal tumor survival within the ER-negative tumor
entity. However, Abd El-Rehim and colleagues [11] and Rakha
and colleagues [20] have suggested that adjustment to ster-
oid hormone receptor expression would not alter the adverse
survival impact of basal phenotype in breast cancer. In our
study the lack of prognostic association was not due to the
method of tumor classification, because the same result was
obtained within the ER-negative subgroup when basal-like
tumors were identified either by IHC or by two different micro-
array-based classifications. These results are in agreement
with the earlier microarray-based prognostic studies, which
indicate that tumors with a basal-like gene expression signa-
ture have a similar prognosis to that of the ERBB2 cluster [7-
9]. It is concluded that all ER-negative tumors can be classified
as having a relatively poor prognosis, irrespective of the cytok-
eratin composition or gene expression signature.
Studies of basal-like breast cancer are likely to be influenced
by the ER status, which is a central factor determining both
prognosis and gene expression patterns [1,2,5,6]. To study
the basal-phenotype breast cancer more specifically without
the influence of ER status, we performed a gene expression
microarray study for ER-negative breast cancers. This enabled
us to look more specifically at the gene expression profile and
clinical behavior of the basal-phenotype tumors when the
impact of information already included in the ER status was
excluded. We were able to separate two tumor clusters, the
basal-like and the non-basal-like, by using a gene set gener-
ated for the basal cytokeratin immunopositive tumors. The
unique gene expression profile found for the CK5/14 immuno-
positive tumors within the ER-negative tumor entity implies that
the basal-like expression profile differed significantly from the
rest of the ER-negative tumors and that this tumor subgroup is
biologically distinct not only in the general breast cancer pop-
ulation but also within ER-negative tumor entity.
Our CK5/14-associated gene signature identified basal-like
tumors within the ER-negative tumor entity very similarly to the
clustering with the intrinsic gene set by Sorlie and colleagues
[7]. Whereas all except one of the CK5/14-positive tumors
were classified to the basal-like cluster with our CK5/14-asso-
ciated genes, four tumors with a CK5/14-positive
immunophenotype were found in the non-basal-like cluster
with Sorlie's intrinsic gene set. This indicates that our top 500
ranked basal genes were better classifiers for CK5/14 IHC
status than Sorlie's intrinsic gene set. This is not surprising
given that our basal gene list was generated for this purpose
and from this very material. Interestingly, all seven CK5/14-
negative tumors categorized into the basal-like cluster by our
basal-associated genes were also found in the basal-like
tumor subgroup when performing the analysis with the intrin-
sic gene set as defined by Sorlie and colleagues. Hence, for
these seven cases the two microarray-analysis-based classifi-
ers agreed on the basal-like status but disagreed with the
CK5/14 immunostaining.
To verify that these tumors had not been misclassified with
regard to basal-like status when using TMAs, we immunos-
tained the entire tumor sections of five of these tumors. Two of
the tumors were scored as CK5/14 positive in entire sections,
indicating that the TMA sampling technique (using tissue
cores with 0.6 mm diameter) leads to the misclassification of
some basal-like tumors in IHC. Expression of basal cytokerat-
ins often shows a high degree of intratumoral heterogeneity
[22], which is likely to explain differences obtained between
TMAs and entire tissue sections. However, even when per-
formed on entire tumor sections, CK5/14 IHC may not recog-
nize all of the basal-like subtype breast cancers as defined by
gene expression profiles. Despite the fact that our gene
expression signature was generated to be specifically associ-
ated with CK5/14 positivity, it clearly also recognizes a distinct
set of CK5/14-negative tumors.
It has previously been suggested that the basal-like tumor type
cluster is most optimally identified by IHC when using a com-
bination of positive CK5/6 and/or EGFR, and negative ER and
HER-2 staining results as classification criteria [23,40]. In
addition, vimentin and c-kit, which have been shown to be
associated with basal cytokeratin immunopositivity along with
EGFR [22,41], have been recognized as good discriminators
for a basal-like expression profile [23,40]. The basal cytokera-
tin-negative tumors that clustered with the basal-like cluster in
this study could be EGFR, vimentin, and/or c-kit-expressing
tumors with a similar gene expression signature to that of basal
cytokeratin-immunopositive breast cancers. It is concluded
that immunohistochemically basal cytokeratin-positive tumors
almost always belong to the basal-like gene expression profile,
but this cluster also includes basal cytokeratin-negative
tumors. Neither a immunohistochemical nor a microarray-
based classification of breast cancers into a basal or non-
basal subgroup is currently considered justified in the clinics,
because direct predictive or prognostic implications are lack-
ing. This could change in the future if differential treatment

Breast Cancer Research Vol 9 No 1 Jumppanen et al.
Page 8 of 10
(page number not for citation purposes)
responsiveness could be confirmed or if treatments specifi-
cally targeting basal-like tumors were developed.
In addition to prognostic assessments, the microarray-based
gene data may be more relevant for revealing the biological
basis of the basal-like tumor classification. For example, the
first genes in the gene list generated for the immunohisto-
chemically predefined CK5/14-positive and ER-negative
tumors included some genes, such as XBP1 and TTF1, that
are known to associate positively with ER status [1,2,6]. These
genes had a significantly lower expression in the basal-like
than in the non-basal-like tumors within the ER-negative tumor
subgroup. It is therefore possible that there are some differ-
ences in the hormone-independence of the basal-like and non-
basal-like tumors within the ER-negative tumor subgroup. In
addition to ER-negativity and poor response to hormone treat-
ment, most basal-like tumors are HER-2 non-amplified. There
are therefore currently no targeted treatment options available
for basal-like breast cancers. Our finding that top signature
genes such as EVA1 (rank 11 and 36), SLC2A1 (rank 42 and
179), and CEACAM1 (rank 148), which are highly expressed
in basal-like tumors and are localized to the cell membrane,
could serve as interesting targets for new drug developments,
similar to the HER-2 oncoprotein in tumors with ERBB2 gene
amplification.
To study the biology of basal-like tumors in more detail and to
evaluate the function of the genes found associated with this
tumor subtype we next found out which biological processes
were enriched in basal-like tumors and used EASE for this pur-
pose. We found that the signature for basal-like tumors was
most significantly enriched for genes associated with epider-
mal differentiation and included the genes encoding CK14
and CK17. Both of these cytokeratins are close partners of
CK5 [42] and have been shown to be expressed in basal-phe-
notype tumors by IHC [11,12,17,20] and by gene expression
microarrays [6,7]. We did not use CK17 in the immunohisto-
chemical determination of basal cytokeratin expression
because we had shown previously that only very few tumors
show CK17 expression in the absence of CK5 and/or CK14
[12]. The biological process of epidermal differentiation may
reflect the basal-phenotype tumor origin. It has been sug-
gested that a CK5/14-positive breast progenitor cell able to
differentiate into both luminal and myoepithelial cells of the
normal breast would be the transformed cell in basal-pheno-
type breast cancer [43,44]. If these cells represent the so-
called cancer stem cell for basal-phenotype breast cancer, the
tumor cells may have the same ability to differentiate as the cell
of origin does. The biological process of development was
fourth in the ranking list and included the EVA1 gene, which
was previously recognized in the basal gene list (rank 11 and
36) as a membrane protein. Other gene ontology terms
enriched in the basal-like gene signature, such as protein and
macromolecular biosynthesis, nuclear division, and M phase,
were indicative of a high proliferation rate. Previous studies
have also associated the basal-like subgroup with a high
expression of genes involved in proliferation [14,22], and our
results suggest that this is true even when compared with the
other subgroups, such as amplified HER-2, within the ER-neg-
ative entity.
Conclusion
Basal cytokeratin immunopositivity predicts early breast can-
cer relapse, and these tumors differ from other ER-negative
breast cancers biologically because they have a distinct gene
expression profile. Despite this, the basal cytokeratin-express-
ing tumors show a similar prognosis to that of non-basal ER-
negative tumors. As regards classification, immunohistochem-
ically basal cytokeratin-positive tumors almost always show a
basal-like gene expression signature. We were able to identify
several immunohistochemically basal cytokeratin-negative
tumors, which have a similar gene expression profile to that of
the basal cytokeratin-immunopositive breast cancers.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
MJ performed and analyzed IHC and CISH stainings from the
TMAs, and drafted and finalized the manuscript. SG per-
formed and analyzed the microarrays and helped in the draft-
ing of the manuscript. Päivikki Kauraniemi helped with the
interpretation of the results and with drafting the manuscript.
MT helped with the finalization of the manuscript. PB per-
formed the statistics for the tables and figures. MK conducted
the analysis of the membrane association of the genes. Pasi
Kataja performed the scanning of the slides for virtual micros-
copy, and ML prepared the final virtual slides for the Internet.
ÅB and MF coordinated the study on their behalf. JI coordi-
nated the study and helped to draft and finalize the manuscript.
All authors read and approved the final manuscript.
Additional files
The following Additional files are available online:
Additional File 1
A PDF file containing a heat map of 50 ER-negative
tumors based on the top 500 gene set generated for the
CK5/14-positive tumors. Yellow indicates the basal-like
cluster and black the non-basal-like cluster.
See http://www.biomedcentral.com/content/
supplementary/bcr1649-S1.pdf

Available online http://breast-cancer-research.com/content/9/1/R16
Page 9 of 10
(page number not for citation purposes)
Acknowledgements
We are grateful to the South Sweden Breast Cancer Group for provid-
ing us with the clinical follow-up data and to the participating depart-
ments for providing us with the samples. We thank Ms Sari Toivola, Ms
Eeva Riikonen, Ms Ritva Kujala, Ms Helvi Salmela, Ms Pirjo Pekkala, and
Ms Päivi Kärki for skillful technical assistance. This study was financially
supported by grants from the Pirkanmaa Hospital District Research
Foundation, the Medical Research Fund of Seinäjoki Central Hospital,
the Swedish Cancer Society, the Swedish Research Council, the Sigrid
Juselius Foundation, Algol-Award, Oy Eli Lilly Finland Ab, and the Finnish
Cancer Foundation.
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Additional File 2
An Excel file containing the top 200 genes list generated
for the immunohistochemically CK5/14-positive ER-
negative breast cancers. The membrane association is
defined.
See http://www.biomedcentral.com/content/
supplementary/bcr1649-S2.xls
Additional File 3
A PDF file containing a heat map of 50 ER-negative
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Additional File 4
A PDF file containing the results of a gene ontology
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See http://www.biomedcentral.com/content/
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