Cancer Therapy Vol 6, page 773

773
Cancer Therapy Vol 6, 773-782, 2008

Molecular prognostic factors: clinical implications
in patients with breast cancer
Review Article

Giuseppe Tonini*, Maria Elisabetta Fratto, Gaia Schiavon
Department of Medical Oncology, University Campus Bio-Medico, Rome
__________________________________________________________________________________
*Correspondence: Giuseppe Tonini MD, PhD., Medical Oncology, University Campus Bio-Medico of Rome, Via Alvaro del Portillo,
200, 00128 Rome, Italy; Tel :0039-06-225411201; Fax: 0039-06-934; e-mail: g.tonini@unicampus.it
Key words: Prognostic factors, molecular classification, breast cancer, gene expression profiling, Oncotype Dx
Abbreviations: cyclophosphamide, methotrexate, and 5-fluorouracil, (CMF); estrogen receptor !, (ER); Microarray for Node-Negative
Disease

may Avoid Chemotherapy, (MINDACT); National Surgical Adjuvant Breast and Bowel Project, (NSABP); plasminogen
activator inhibitor, (PAI-1); Recurrence Score, (RS); urokinase plasminogen activator, (uPA)

Received: 17 April 2008; Revised: 15 July 2008
Accepted: 4 August 2008; electronically published: October 2008

Summary
Many molecular markers have been recently identified as prognostic and predictive factors in patients with breast
cancer. Histological type, grading, tumor size, lymph node involvement, and estrogen receptor ! (ER) and HER-2
receptor status all influence prognosis and the probability of response to systemic therapies. Among other
prognostic factors, p53 mutations and Bcl-2 amplification are associated with higher probability of relapse or death.
Recently, proteomic and gene-expression profiling methods are being explored to quantify the expression of
multiple genes and combine the gene expression measurements into prediction scores that may foretell clinical
outcome. Among new methods that can measure ER mRNA expression, Oncotype DX represents an important
advance in the diagnosis of ER-positive breast cancers. Several reports have confirmed the prognostic value of this
diagnostic test. Among other multigene prediction scores, the most promising is the Amsterdam Signature, in fact
an on-going trial is evaluating this test to stratify patients that need chemotherapy or not (MINDACT trial). Gene
profiling has also been used to predict metastasis to distant organs. Thanks to gene-expression profiling
technologies, different molecular subtypes of breast cancer associated with different natural histories have been
identified. In fact many studies have shown that the prognosis and chemotherapy sensitivity of the different
molecular subgroups are different. Thanks to these new technologies, new perspectives are opening. Trials based on
genetic profiling will serve as an important resource for evaluating new molecular signatures providing a novel,
personalized dimension for the treatment and care of breast cancer patients. Finally further studies are needed to
improve current prognostic and treatment predictive tools.


I. Introduction
Breast cancer is an heterogeneous disease and the
existing classifications are not fully associated with the
varied clinical course of this disease. Histological type,
grading, tumor size, lymph node involvement, estrogen
receptor ! (ER) and HER-2 receptor status all influence
prognosis and the probability of response to systemic
therapies, having also a predictive value. These clinical
variables can be combined into multivariate outcome
prediction models, as The Nottingham Prognostic Index
and Adjuvant! Model for early breast cancer (DEredita et
al, 2001; Olivotto et al, 2005). However, the substantial
variability in disease outcome within each risk category
means that the outcome prediction models used do not
include all the possible variables of patients with breast
cancer. The different clinical course of patients with
histologically identical tumors may be the result of
molecular differences among cancers. So detailed
molecular analysis of the cancer could give information on
prognosis and prediction. Recently, proteomic and gene-
expression profiling methods are being explored as
diagnostic tools. These tests quantify the expression of
multiple genes and combine the gene expression
measurements into prediction scores that may foretell
clinical outcome more accurately than any of the genes
alone (Pusztai et al, 2006).

Tonini et al: Molecular prognostic factors: clinical implications in patients with breast cancer
774
II. Traditional prognostic and
predictive factors in breast cancer
Traditionally, prognostic factors provide prospective
information about overall patient prognosis, while
predictive factors provide information about the chance of
patient response to treatment. Some markers may be both
prognostic and predictive. Moreover, a positive prognostic
factor may have a negative predictive value and vice
versa, so accurate studies are needed to understand the
right strategy for patients with breast cancer.

A. Molecular markers as prognostic
factors
The most significant prognostic factor for patients
with

breast cancer is the presence or absence of axillary

lymph node involvement. Furthermore, there is a direct
relationship

between the number of involved axillary
nodes and the risk for

distant recurrence, so patients with
node-negative status (including the sentinel-node-negative
classification) are patients with low-risk disease (Early
Breast Cancer Trialists Collaborative Group (EBCTCG), 2005).
However, the St Gallen (Cody et al, 2004) treatment
guidelines were updated in 2005 to include the
intermediate-risk category, so the presence of positive
axillary nodes in the absence of other high-risk
characteristics no longer defines high-risk disease (Cody et
al, 2004). Other new adverse prognostic factors were also
accepted: HER2/ neu overexpression, grading, tumor size,
peritumoral vascular invasion and age of the patients.
Table 1 shows the risk categories for patients with
operable breast cancer established at the 2005 St Gallen
meeting. Combining these factors it is possible divide
patients in three categories: low, intermediate and high
risk. According to the category it is possible to make a
choice regarding the right treatment strategy. Although
several novel prognostic factors, including proliferation
markers, such as S-phase fraction and Ki-67, lymph node
micrometastases (Truong et al, 2005, urokinase plasminogen
activator (uPA)/plasminogen activator inhibitor (PAI-1)
system expression (Look et al, 2002), cyclin-E
overexpression have been proposed, more studies are
needed to validate these markers. A recent meta-analysis
showed the prognostic value of Ki-67 in early breast
cancer. 68 studies were identified and 46 studies including
12 155 patients were considered for evaluation of
correlation between KI-67/ MIB-1 + and Disease-Free
Survival and Overall Survival. Ki-67/MIB-1 + resulted
associated with higher probability of relapse (P<0.001)
and worse survival (P<0.001) in all patients, independently
from nodal status (De Azambuja et al, 2007) uPA/PAI-1
system is associated with invasion, angiogenesis, and
metastasis in preclinical models (Stephens et al, 1998).
Several studies have demonstrated that the overexpression
of uPA and/or PAI-1 have been related to poor prognosis
in early-stage breast cancer. In fact patients
overexpressing these markers have higher risk of
recurrence and death (Bouchet et al, 1994; Harbeck et al,
2006). Moreover, studies in early breast cancer patients
suggest that these two markers are very strong prognostic
factors, independently of size, grade, and hormone
receptor status (Look et al, 2002). The first interim report of
a prospective trial using uPA and PAI-1 levels to stratify
node-negative patients has been published (Jnicke et al,
2001). Among the 556 patients accrued, those patients
whose tumors expressed low levels of both markers were
not treated with adjuvant chemotherapy. Patients whose
tumors showed elevated uPA and/or PAI-1 levels were
randomly assigned to adjuvant chemotherapy or no
adjuvant chemotherapy. The estimated 3-year recurrence
rate for 241 patients with low levels of both uPA/PAI-1
was 6.7%, while the recurrence rate for patients with
elevated uPA and/or PAI-1 levels who did not receive
chemotherapy was double. Another prognostic factor for
patients with breast cancer is cyclin E. Elevated levels of
cyclin E protein have been associated with a poor
prognosis in breast cancer in some studies. In a recent
meta-analysis of cyclin E overexpression of 2,534 patients
in 12 trials, the overexpression of cyclin E was associated
with a 1.72-fold increased risk of recurrence in
multivariate analysis (Wang and Shao, 2006).


Table 1. Risk categories based on disease/patient characteristics for women with early breast cancer.

LOW RISK INTERMEDIATE RISK HIGH RISK
Node-negative and all these
characteristics:
T</= 2 cm
G1
No vascular invasion
HER-2 negative
Age>/= 35years
Node-negative and at least one of
these characteristics:
T > 2 cm
G2-3
Vascular invasion
HER-2 positive
Age< 35years
Node-positive (1-3) and HER-2
negative
Node-positive (1-3) and HER-2
positive
Node-positive (>/=4)

Cancer Therapy Vol 6, page 775
775
However, conclusions regarding the prognostic value of
cyclin E in the published literature are mixed and
additional properly designed studies are required to
evaluate whether this marker has clinical utility, especially
in the setting of no adjuvant chemotherapy. Finally, Harris
et al. published the American Society of Clinical
Oncology 2007 Update of recommendations for the use of
tumor markers in breast cancer. Among the thirteen
categories considered, CA 15-3, CA 27.29,
carcinoembryonic antigen, estrogen receptor, progesterone
receptor, human epidermal growth factor receptor 2,
urokinase plasminogen activator, plasminogen activator
inhibitor 1 showed evidence of clinical utility and were
recommended. On the contrary DNA/ploidy by flow
cytometry, p53, cathepsin D, cyclin E, proteomics, certain
multiparameter assays, detection of bone marrow
micrometastases and circulating tumor cells demonstrated
insufficient evidence to support routine use in clinical
practice (Harris et al, 2007). Moreover, Mc Shane and
colleagues suggested guidelines to provide information
about the tumor markers studies, in fact the results
presented for each tumor marker often are in contradiction.
The goal of these guidelines is to evaluate the studies,
understand the usefulness of the data, assessing the value
of the conclusions (McShane et al, 2006). Concluding, more
than 100 individual factors have been investigated and
reported in the literature. However, few of these factors
have found their way into clinical application as
prognostic tools, or contributed greatly to understanding of
tumor biology.

B. Molecular markers as predictive
factors of response/resistance to
chemotherapy
Chemoresistance is the main obstacle to successful
therapy in cancer patients. When evaluating a new
prognostic factor, its potential predictive role should be
considered. In fact, a negative prognostic factor may have
a positive predictive value. For example p53 mutations
and HER-2 overexpression identify patients with a poor
prognosis (Carreo et al, 2002), both are also associated
with (although not fully predictive for) responsiveness to
specific treatments (Geisler et al, 2001). Moreover, even if
the identification of reliable predictive factors has the
potential to spare patients from ineffective treatments and
unnecessary side effects, it is difficult to find a factor that
may guarantee therapeutic success. For example, while ER
negativity is associated with lack of response to endocrine
therapy, not all patients with ER+ tumors may benefit
from such therapy. However, for the first time at the 2005
St Gallen meeting, endocrine responsiveness was
identified as the primary consideration when making
treatment decision, instead of the risk category (Goldhirsch
et al, 2005). Moreover, the 2007 St Gallen meeting
underlined the importance of the molecular assessment of
tumor biology. For those with a highly endocrine-
responsive tumor, for example, the priority is to select the
best possible hormonal therapy with the possibility of
adding chemotherapy. For those with an endocrine non-
responsive tumor, the priority is the best possible
chemotherapy regimen. Similarly, while the absence of
HER2/neu overexpression has been established as a
predictive factor for non-responsiveness to trastuzumab
therapy, not all HER2/neu-overexpressing tumors are
trastuzumab sensitive (Tokunaga et al, 2006), reflecting the
complexity of breast cancer genetics. Another problem is
to evaluate if a predictive factor is a causal factor or just a
co-variate. In fact whereas early studies suggested that
HER2 overexpression predict for sensitivity to
anthracyclines (Paik et al, 1998), recent studies have shown
that TOPO-IIa, not HER2, overexpression predicts for
anthracycline sensitivity in tumors with coamplification of
the two genes (Scandinavian Breast Group Trial 9401, 2006).
Regarding mutations in P53 gene, it has also been shown
to correlate with chemosensitivity in patients with breast
cancer. Specifically, responsiveness to anthracycline- or
mitomycin-containing chemotherapy is reduced by
defective P53 status. In contrast, the efficacy of paclitaxel
seems to be independent of p53 expression. Geisler and
colleagues showed in 2003 that mutations in the P53 gene,
in particular those affecting or disrupting the loop domains
L2 or L3 of the p53 protein, were associated with lack of
response to chemotherapy (P=0.063 for all mutations and
P=0.008 for mutations affecting L2/L3, respectively).
Similarly, the overexpression of HER-2 (P = 0.041), a
high histological grade (P=0.023) and lack of expression
of bcl-2 (P=0.018) predicted chemoresistance to
doxorubicin (Geisler et al, 2001). These results were
confirmed by Di Leo and colleagues in 2007 who
evaluated in 108 patients with metastatic breast cancer
treated with doxorubicin or docetaxel. P53 gene mutations
were observed in 20% of patients. In patients with a
mutated p53, a lower percentage of responders was
observed in the doxorubicin arm (17% VS 27%),
compared with the docetaxel arm (50% VS 36%). So p53
gene mutations compromised the efficacy of doxorubicin,
not interfering with the antitumor activity of docetaxel (Di
Leo et al, 2007). However, available data from trials do not
support the use of tumor P53 status when selecting
patients for a given treatment. Another study evaluated the
prognostic relevance of a novel semiquantitative
classification of Bcl2 immunohistochemical expression in
breast cancer. Bcl-2 expression was evaluated in 442
patients, resulting as independent predictor of clinical
outcome in both node-negative and node-positive patients.
In fact patients with Bcl-2 negative immunostaining had
higher probability of relapse (5 times) or death (7 times)
(Trer et al, 2007) Thus, more research is necessary before
BCL-2 status and other new prognostic factors can be
accepted as a predictive tool for breast cancer therapy.

III. Genomic profile for prognosis and
prediction
Array-based molecular profiling represents a
technological advance in how tumor samples can be
analyzed. In fact microarray technology allows the
simultaneous analysis of many thousands of individual
genes in cell or tissue samples, so the complex biology of
cancer may be studied much more comprehensively than
previously possible (Gruvberger-Saal et al, 2006). In recent
years, microarrays have been used extensively to study
molecular differences among different types of cancer.
Tonini et al: Molecular prognostic factors: clinical implications in patients with breast cancer
776
One of the most studied tumor types is breast cancer,
because of its complexity and the different behaviour of
the same histological tumors. Relationships between gene
expression and distant metastasis have been identified and
characteristic patterns have emerged, reflecting molecular
differences between tumors. This has allowed the
identification of different subtypes of breast cancer based
on gene profiling. These molecular differences have been
shown to correlate with clinical features, such as survival,
prognosis and treatment sensitivity, as well as traditional
histopathological parameters.

A. Gene profiling and distant metastasis
Gene profiling has been used to predict metastasis to
distant organs. In fact genes associated with bone
metastases were identified in experimental models (Kang et
al, 2003). In humans, Smid et al. identified a 69-gene panel
associated with bone metastasis in patients with node
negative breast cancer. TFF1 was the most differentially
expressed gene in an independent patient cohort (p =
0.0015). A classifier of 31 genes was identified, which in
an independent validation set predicted all tumors
relapsing to bone with a specificity of 50% (Smid et al,
2006). Moreover, Minn and colleagues identified in 2005 a
set of genes that mediates breast cancer metastasis to the
lungs. In fact they found significant differences in lung-
metastases-free survival between patients with or without
expression of lung metastasis gene signature, especially in
patients with poor prognosis (p= 0,008) or ER-negative
(p=0,004) (Minn et al, 2005). Many studies are on-going to
find other genes related to a specific site of metastasis,
giving a contribution for clinical research and then clinical
application.

B. Role of Oncotype Gene Recurrence
Score (RS) assay, Amsterdam and Rotterdam
signature in prognosis and prediction
Determination of ER status is essential to determine
whether a patient is a candidate for endocrine therapy.
This test is routinely used in the clinic, but the existing
immunohistochemical assays have only modest positive
predictive value (30%-60%) for response to hormonal
therapies (Bonneterre et al, 2000; Mouridsen et al, 2001)
Furthermore, there are intra- and interlaboratory variation
in ER results because of differences among laboratories
regarding fixation, antigen retrieval and staining methods
(Rhodes et al, 2000). So more accurate and more reliable
predictors of benefit from hormonal therapy based on
gene-profiling are developing and are necessary for a
greater accuracy in the treatment strategy for patients with
hormone-responsive breast cancer. Oncotype DX
(Genomic Health Inc., Redwood City, CA) represents an
important advance in the diagnosis of ER-positive breast
cancers (Figure 1). This RT-PCR-based assay measures
ER mRNA expression in a highly quantitative and
reproducible manner and the expression of several
downstream ER-regulated genes (PR, BCL-2, SCUBE-2)
that may contain information on ER functionality..





Figure 1. Oncotype DX Gene Recurrence Score Assay: This test evaluates the expression of 16 cancer genes and 5 Reference Genes (A)
providing a Recurrence Score from 0 to 100 (B). Thanks to this score, population is stratified in 3 risk categories (C), useful to
understand patient outcomes and the potential benefit of chemotherapy.
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777
The same assay also quantifies HER-2 expression,
proliferation- and invasion-related genes. Combining
information from each of these measurements into a
prediction score can give important information on patient
prognosis. A study examined the correlation between the
Oncotype DX recurrence score and the likelihood of
distant relapse in 668 ER-positive, node-negative,
tamoxifen-treated patients who were enrolled in the
National Surgical Adjuvant Breast and Bowel Project
(NSABP) clinical trial B14. 51%, 22%, and 27% of these
ER-positive patients were categorized as low,
intermediate, and high risk for recurrence after tamoxifen
therapy, respectively. The observed 10-year distant
recurrence rates were 6.8%, 14.3%, and 30.5% in the three
risk categories, respectively (p < 0.001). In a multivariate
analysis, the recurrence score predicted relapse and overall
survival independently of age and tumor size (Paik et al,
2004). A recent report examined the value of the
recurrence score for predicting benefit from adjuvant
cyclophosphamide, methotrexate, and 5-fluorouracil
(CMF) chemotherapy in 651 patients with ER-positive,
node-negative breast cancer included in the NSABP B20
randomized study. Higher recurrence scores were
associated with greater benefit from adjuvant CMF
chemotherapy (p = 0.038). The hazard ratio for distant
recurrence after CMF chemotherapy was 1.31 for patients
with a recurrence score <18 and 0.26 for patients with a
recurrence score >31. The absolute improvement in 10-
year distant recurrence-free survival was 28% (60% vs.
88%) in patients with a recurrence score >31, while there
was no benefit in patients with a recurrence score <18
(Paik et al, 2006). These data indicate that a high recurrence
score could identify a subset of women with ER-positive
and node-negative breast cancer that have a high risk for
recurrence with tamoxifen therapy alone and that this risk
can be reduced with the administration of adjuvant CMF
chemotherapy. Several other multigene prediction scores
have been developed in order to improve the predictive
value of ER and better estimate who will benefit of
endocrine therapy. In one study, 81 genes were found to
be differentially expressed between tamoxifen-sensitive
and tamoxifen-resistant, ER-positive breast cancers (n =
46) and a 44-gene predictive signature was developed.
This signature was tested on 66 independent ER-positive
cases and could select patients who had prolonged
progression-free survival (Jansen et al, 2005). Another
prediction score is the Amsterdam Signature
(Mammaprint !), a DNA microarray based test that
measures the activity of 70 genes to define a prognostic
profile. This gene profiling was developed from tumors of
women under the age of 55 years-old with Stage I or II
breast cancer to identify genes associated with bad or good
prognosis (van de Vijver et al, 2002). The Microarray for
Node-Negative Disease

may Avoid Chemotherapy
(MINDACT) trial has been designed to

compare the ability
of the 70-gene prognostic profile versus clinical

and
pathological criteria to identify women with node-negative

breast cancer who are unlikely to benefit from adjuvant
chemotherapy.

Enrollment of 6000 patients is planned, the
test will be performed to assign patients to endocrine
therapy or chemotherapy (Rutgers, 2007). Figure 2 shows
the study design of this trial. Moreover, Foekens and
colleagues evaluated a 76-gene prognostic signature



Figure 2. Study design of the Breast International Group 1 MINDACT Trial. Abbreviations: CT: chemotherapy; HT: Hormonotherapy

Tonini et al: Molecular prognostic factors: clinical implications in patients with breast cancer
778
(Rotterdam signature) in lymph node-negative breast
cancer patients. This prognostic tool was highly
informative in identifying patients with distant metastasis
within 5 years, even when corrected for traditional
prognostic factors in multivariate analysis. The 5- and 10-
year distant metastasis-free survival were 96% and 94%
for the good profile group and 74% and 65% for the poor
profile group (Foekens et al, 2006). Once validated, these
prognostic signatures could be considered as a prognostic
and predictive test with relevant clinical utility.

IV. Prognostic value of the new
classification for breast cancer
Different molecular subtypes of breast cancer
associated with different natural histories were identified
thanks to the advent of gene-expression profiling
technologies. Moreover, new therapeutic targets and
treatments that are effective in particular molecular subsets
have been tested so that each patient will be treated only
with the drugs really effective. Thanks to microarray
technology, investigators determined that there were breast
cancer subtypes with distinct gene expression patterns and
different prognoses (Sotiriou et al, 2003) that persisted in
primary breast cancers as well as their metastases (Weigelt
et al, 2003). The first study to examine comprehensively
gene-expression patterns of breast cancer suggested that at
least four major molecular classes of breast cancer exist:
luminal-like, basal-like, normal-like, and HER-2 positive
(Perou et al, 2000). Other studies using gene expression
profiling revealed that within the ER-positive tumors at
least two subtypes, luminal A and luminal B, with
important differences in gene expression and prognosis
could be distinguished. On the contrary, hormone
receptor-negative breast cancer comprised two distinct
subtypes, the HER2 subtype and the basal-like subtype.
These subtypes differ in biology and behaviour and are
associated with a poor outcome (Pusztai et al, 2003).
Although most investigators have identified also other
subtypes, these studies have been performed on relatively
small datasets, so the number of different breast cancer
subtypes is still unknown. In addition to the biological
implications, the identification of different breast cancer
subtypes is also associated with different prognostic
significance. A small overview of the different cancer
subtypes is described in the next paragraphs

(Srlie et al,
2003).

A. Luminal subtypes: Expression
patterns, clinical features and prognosis
The luminal subtypes represent the hormone
receptor-positive breast cancers and have expression
patterns similar to the luminal epithelial component of the
breast (Perou et al, 2000). These patterns include expression
of ER, genes associated with ER activation and luminal
cytokeratins 8/18. These tumors are often grade I and
often do not have p53 mutations (less than 20%). At least
two subtypes of the luminal cluster exist, luminal A and
luminal B. Although both are hormone receptor positive,
these two luminal subtypes have different characteristics.
In fact luminal A usually has higher expression of ER-
related genes and lower expression of proliferative genes
than luminal B
40
. Luminal breast cancers are the most
common subtype of breast cancer, in fact they represent at
least 67% of the tumors (Carey et al, 2004). In general, the
luminal subtypes have a good prognosis, even if luminal B
carry a significantly worse prognosis than luminal A in
multiple data sets (Pusztai et al, 2003). Part of this different
outcome may be due to variations in response to treatment.
Luminal breast cancers are treated with hormone therapy.
In fact several studies have demonstrated that ER-positive
tumors respond poorly to conventional chemotherapy
(Rouzier et al, 2005) Data from Oncotype DX Score suggest
that tumors with low Recurrence Scores are luminal A
whereas those with high Recurrence Scores are luminal B.
Thus, it appears that luminal A tumors may be adequately
treated with endocrine therapy alone, whereas the more
proliferative luminal B tumors may be those that benefit
from chemotherapy added to endocrine therapy (Paik et al,
2004). Among targeted therapies active in luminal
subtypes, the anti-vascular endothelial growth factor
antibody bevacizumab has recently shown to improve
survival in metastatic breast cancer when combined with
paclitaxel (Miller et al, 2005). Interestingly, more than 60%
of the patients in this trial were hormone receptor-positive
and almost none was HER2-positive, suggesting that
bevacizumab may be particularly effective in the luminal
subtypes.

B. HER-2 subtype: Expression patterns,
clinical features and prognosis
The HER2 subtype refers to the large group of
hormone receptor-negative tumors identified by gene
expression array. HER2-positive tumors clinically refer to
those identified by immunostaining for HER2
overexpression or FISH for excess gene copy number,
even if there is not a perfect correspondence between the
clinically evaluated HER2-positive and the HER2-array
subtype. However, tumors that are both hormone-receptor
positive and HER2 positive by immunohistochemistry or
FISH often are included in the luminal subtypes rather
than the HER2-array subtype. The HER2-array tumors are
characterized by overexpression of other genes in the
ERBB2 amplification such as GRB7. Like basal-like
tumors, the HER2-array subtype tumors have often p53
mutations (40%-80%) and are significantly more likely to
be grade 3 (P=0.0002) than luminal A tumors (Srlie et al,
2001). There is no association of the HER2-array subtype
with age, race or known risk factors (Perou et al, 2000).
Moreover, HER2-array subtype tumors are often lymph-
nodes positive, more than two-fold than luminal A tumors.
Despite its poor prognosis, the HER2-array subtype has
also demonstrated sensitivity to anthracycline and taxane-
based neoadjuvant chemotherapy, with significantly
higher pathologic complete response than luminal breast
tumors (46% v 7%; P<0.001) (Carey et al, 2004). Like the
other ER-negative subtype, the basal-like, the HER2-array
subtypes poor prognosis seems to derive from a higher
risk of early relapse because of an incomplete eradication
of tumor cells (Carey et al, 200). In recent trials, the benefits
of each generation of increasing chemotherapy
aggressiveness have been demonstrated in ER-negative
Cancer Therapy Vol 6, page 779
779
breast cancer. In fact the evaluation of the efficacy of
increased anthracycline dose and intensity (CALGB
8541), of taxane added to anthracycline (CALGB 9344)
and of increased dose density (CALGB 9741) were
demonstrated in the ER-negative subset of patients (Berry
et al, 2004). This suggests that the HER2 and basal-like
subtypes (the majority of ER-negative tumors) are the
tumors that derive the most benefit from improvements in
chemotherapy. However, the treatment for the HER2-array
subtype includes targeted agents, including the anti-HER2
monoclonal antibody trastuzumab. The effectiveness of
trastuzumab in metastatic breast cancer and in the adjuvant
setting supports the idea that this strategy is really
effective (Perez et al, 2005; Romond et al, 2005), even if not
all HER2-positive tumors respond to trastuzumab. PTEN
loss or abrogation (Nagata et al, 2004) and CXCR4
upregulation (Tripathy et al, 2005) have been implicated in
trastuzumab resistance and may provide targets for
combination strategies that could give better results in the
future.

C. Basal-like subtype: Expression
patterns, clinical features and prognosis
The basal-like subtype of breast cancer has
expression patterns similar to the basal epithelial cells of
other parts of the body and to the normal breast
myoepithelial cells. This subtype does not express ER and
related genes, has a low expression of HER2, a strong
expression of basal cytokeratins 5, 6 and 17 and
proliferation-related genes (Weigelt et al, 2003). This
subtype is also characterized by low expression of BRCA1
(Abd et al, 2005). Basal-like tumors often have aggressive
features such as p53 mutations and are significantly more
likely to be grade 3(P < 0.0001) than luminal A tumors
(Miller et al, 2005). Basal-like tumors represent 20% of the
tumors and are the most common among premenopausal
African American women (39%) compared with
postmenopausal African American (14%) or non-African
American women of any age (16%) (P= 0.0001) (Perou et
al, 2000). This subtype is also associated with some risk
factors, in fact most women with BRCA1 mutations
generally develop basal-like breast cancer (Turner et al,
2004). So basal-like breast cancer has a poor prognosis
(Carey et al, 2004)
,
but it is not clear if it is due to poor
therapy options and/or the biological aggressiveness. In
fact basal-like breast cancer can not be treated with
conventional targeted therapies for breast cancer such as
endocrine therapy or trastuzumab because of their triple-
negative receptor status (ER, PR, and HER2).
Chemotherapy is the only option for this setting of
patients, in fact basal-like breast cancers are sensitive to
conventional chemotherapy. In two studies (Srlie et al,
2001; Rouzier et al, 2004), response to anthracycline-based
or combination anthracycline and taxane-based
neoadjuvant chemotherapy among basal-like tumors was
evaluated, with higher response rates among basal-like
breast cancers than non-basal like. These studies suggest
that the poor prognosis of patients with basal-like tumors
is not due to the initial chemoresistance, but it reflects the
fewer treatment options available for ER-, PR- and HER2-
negative tumors and/or to the intrinsic aggressiveness of
this subtype. The only targeted option evaluated in this
subtype is the treatment with epidermal growth factor
receptor-inhibitors (Srlie et al, 2001). This hypothesis is
being tested in several preclinical and clinical trials.
Finally, Cheang MG et al showed that basal-like breast
cancer defined by five biomarkers has superior prognostic
value than triple-negative phenotype. In fact, EGFR and
cytokeratin 5/6 are markers of basal-like breast cancer
applicable to standard pathology specimens that can be
added to the estrogen receptor, progesterone receptor and
HER-2 markers. Among the 3,744 patients with invasive
breast cancer, 17% were basal using the triple-negative
definition and 9% were basal using the five-marker
method. They demonstrated that the five-marker panel is
significantly more prognostic than the three-marker panel.
In fact among triple-negative patients treated with
adjuvant anthracycline-based chemotherapy, the additional
positive basal markers identified a cohort of patients with
significantly worse outcome (Carey et al, 2007).

V. Future directions
Current breast cancer treatment guidelines are based
on the results of randomized clinical trials in defined
patient populations affected by tumors with different
biological characteristics. Anyway, it is clear that better
prognostic and predictive factors are needed for the choice
of treatment strategies individualized for each patient.
Considering molecular prognostic and predictive factors,
gene profiling seems promising, although further
validation is mandatory. In fact, only small studies in
patients with early breast cancer patients have been
performed. Given the high potential of gene expression
profiling to change clinical practice, it is now important to
validate this new prognostic tool in large, independent and
prospective trials. In fact only large, well-conducted,
biologically-based prospective trials will allow us to reach
the needed conclusions and to shorten the time for the
clinical application of these new markers and techniques.
Nevertheless, encouraging results from studies using
microarrays for identification of genes related to cancer
prognosis and prediction indicate that this technique will
provide soon a novel, personalized dimension for the
treatment and care of breast cancer patients. These studies
have also

identified genes whose protein products may
provide therapeutic

targets for the progressive
development of novel, more effective

and less toxic
chemotherapeutic agents. Some studies have also
identified a group of patients with good prognosis, who
may not benefit from adjuvant systemic chemotherapy. So
further studies are needed to understand the effects of
different treatment regimens on disease outcome and/or
molecular subtype to identify those patients who most
likely could benefit from adjuvant or neoadjuvant
treatment regimens. In addition, microarrays provide an
opportunity to potentially identify new targets for therapy,
in fact it will be important to develop targeted therapies,
especially for patients with aggressive tumors.
Concluding, the next challenge for researchers is try to
translate all the results of preclinical studies in clinical
setting to select only the drugs really effective for each
patient.
Tonini et al: Molecular prognostic factors: clinical implications in patients with breast cancer
780
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Giuseppe Tonini