Breast cancer is a genetically and clinically heterogeneous disease.1 [628665]

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Introduction
Breast cancer is a genetically and clinically heterogeneous disease.1
In order to organize this heterogeneity and standardize the lan –
guage, breast cancer classification systems have been developed.
These classification schemes have evolved over many decades into a tool that is used to aid in treatment and prognosis. However, with recent advances in cancer research and an increased molecu –
lar understanding of breast cancer heterogeneity, the current clin –
ical model for breast cancer classification may benefit from the addition of several factors. The identification of tumor initiating cancer stem cells and the five molecular subtypes of breast cancer
*Correspondence to: Vimla Band; Email: [anonimizat]
Submitted: 08/26/10; Accepted: 10/06/10Previously published online:www.landesbioscience.com/journals/cbt/article/13879DOI: 10.4161/cbt.10.10.13879increased understanding of the molecular heterogeneity that
is intrinsic to the various subtypes of breast cancer will likely
shape the future of breast cancer diagnosis, prognosis and
treatment. Advances in the field over the last several decades
have been remarkable and have clearly translated into better patient care as evidenced by the earlier detection, better prognosis and new targeted therapies. There have been two
recent advances in the breast cancer research field that have
lead to paradigm shifts: first, the identification of intrinsic breast tumor subtypes, which has changed the way we think about breast cancer and second, the recent characterization of cancer stem cells (CSCs), which are suspected to be
responsible for tumor initiation, recurrence and resistance to
therapy. These findings have opened new exciting avenues to think about breast cancer therapeutic strategies. w hile
these advances constitute major paradigm shifts within the
research realm, the clinical arena has yet to adopt and apply
our understanding of the molecular basis of the disease to early diagnosis, prognosis and therapy of breast cancers. Here, we will review the current clinical approach to classification of breast cancers, newer molecular-based classification schemes
and potential future of biomarkers representing a functional
classification of breast cancer.Histological, molecular and functional subtypes
of breast cancers
Gautam K. Malhotra,1 Xiangshan Zhao,1 Hamid Band1,2 and v imla Band1,2,*
1Departments of Genetics, Cell Biology and Anatomy; College of Medicine; 2eppley i nstitute for Cancer and Allied Diseases and UNMC- eppley Cancer Center;
University of Nebraska Medical Center; Omaha, N e USA
Key words: breast cancer, tumor heterogeneity, cancer subtypes, stem cells
will help provide an even more comprehensive and clinically rel –
evant classification of breast cancer heterogeneity.
Histological Classifications
of Breast Cancer Subtypes
Unlike colon cancers, defining the progression of breast cancer
has not been possible due to lack of markers that define hyperpla –
sia (typical and atypical), carcinoma in situ and invasive cancer.1
However, breast cancer can be broadly categorized into in situ carcinoma and invasive (infiltrating) carcinoma. Breast carci –
noma in situ is further sub-classified as either ductal or lobu –
lar; growth patterns and cytological features form the basis to distinguish between the two types. Ductal carcinoma in situ (DCIS) is considerably more common than its lobular carcinoma in situ (LCIS) counterpart and encompasses a heterogeneous group of tumors. DCIS has traditionally been further sub- classified based on the architectural features of the tumor which has given rise to five well recognized subtypes: Comedo, Cribiform, Micropapillary, Papillary and Solid ( Fig. 1 ).
2 While
this classification scheme has been a valuable tool for several decades, it relies solely on histology without utilizing newer molecular markers that have a proven prognostic significance.
In light of surgical advances leading to breast-conserving
therapy, it has become necessary to more accurately stratify patients based on relative risk of recurrence or progression. These demands have led to the generation of several newer classification systems that incorporate molecular markers such as ER, PR, ErbB2 (Her2/neu) and p53.
3-6 While the routine
use of these markers for DCIS has not been accepted by the larger medical community, it is notable that the National Comprehensive Cancer Network has included determination of ER status as part of their DCIS workup.
7 This paradigm shift
foreshadows the future of molecular medicine that we have only recently begun to appreciate.
Similar to in situ carcinomas, invasive carcinomas are a het –
erogeneous group of tumors differentiated into histological sub –
types. The major invasive tumor types include infiltrating ductal, invasive lobular, ductal/lobular, mucinous (colloid), tubular, medullary and papillary carcinomas ( Fig. 1 ). Of these, infiltrat –
ing ductal carcinoma (IDC) is, by far, the most common subtype accounting for 70–80% of all invasive lesions.
8 IDC is further
sub-classified as either well-differentiated (grade 1), moderately differentiated (grade 2) or poorly differentiated (grade 3) based

956 Cancer Biology & Therapy volume 10 issue 10
therapies. The recent identification of the molecular subtypes
of breast cancer has begun to address this issue. Recent studies identified several intrinsic molecular subtypes of breast cancer that were later confirmed and classified as: basal-like, ErbB2
+,
normal breast like, luminal subtype A and luminal subtype B
(Fig. 2 ).14-16 More recently, a new subtype classified as “claudin-
low” has also been identified.17,18 These molecular subtypes of
cancer were identified by microarray-based gene expression anal –
ysis and unbiased hierarchical clustering. Notably, the molecular subtypes display highly significant differences in prediction of overall survival, as well as disease-free survival with the basal-like/triple-negative (ER
-/PR-/ErbB2-) subtype having the shortest
survival.15,16 Furthermore, this molecular classification was able
to stratify the ER+ population into several subtypes that, again,
demonstrated a difference in patient survival. This is significant because even though clinical assessment of IDC utilizes ER, PR and ErbB2 status, these markers did not allow separation of the two distinct ER
+ subtypes (i.e., Luminal A and Luminal B) that
have very different clinical outcomes.15,16
The utility of this new molecular classification to predict out –
comes has raised hopes of its adaptation in clinical practice; how –
ever, routine use of microarray analysis or genome sequencing is on the levels of nuclear pleomorphism, glandular/tubule forma –
tion and mitotic index.9
In contrast to DCIS, where the use of molecular markers is still
debated, the utility of ER, PR and HER2/neu is well accepted for IDC and it is recommended that their status be determined on all invasive carcinomas.
7,10 Furthermore, the College of American
Pathologists acknowledges, but does not require or recommend, the use of other ancillary tests (e.g., gene array profiling or immu –
nohistochemical staining for markers other than ER, PR and HER2/neu) as long as sufficient tissue is available.
9 The use of
ER, PR and HER2/neu determination in IDC exemplifies the potential of molecular biomarkers in guiding clinical decisions.
11
Already, the status of these markers helps determine which patients are likely to respond to targeted therapies (i.e., tamoxifen or aromatase inhibitors for ER
+/PR+ patients and trastuzumab or
lapatinib for HER2/neu patients).12,13
Molecular Classifications of Breast Cancer Subtypes
While the current model for breast cancer classification has prog –
nostic value, lack of a molecular component to the classification scheme limits the ability to predict a response to newer targeted Figure 1. Histological classification of breast cancer subtypes. This scheme, currently used by clinicians, categorizes the heterogeneity found in breast
cancer based on architectural features and growth patterns. HPF: high power field.

www.landesbioscience.com Cancer Biology & Therapy 957
of breast cancer, yet another emerging area of intense research
has been that of breast cancer stem cells (CSCs).21 Although the
concept of CSCs is not recent, it has gained significant attention of late due to the isolation and characterization of CSCs as tumor initiating cells in several common malignancies. The cancer stem cell theory simply states that, within a tumor, there exists a small subset of cells that is responsible for tumor initiation and progression, while the remaining bulk of the tumor cells is of low tumorigenic potential.
1 The cell of origin for CSCs is still
undetermined but two prevailing hypotheses propose that CSCs either originate from normal cells within the stem cell hierarchy (Fig. 3 a) or arise from a common normal stem cell ( Fig. 3b ).
Due to the rapid growth of the field, there is considerable con –
fusion regarding the appropriate methods of isolation and identi –
fication of CSCs. Particularly, there are no conclusive markers to differentiate between stem and non-committed progenitor cells. The cellular hierarchy of the mammary gland is not as clearly delineated as that of the hematopoietic system. Several labora –
tories have independently identified markers to isolate CSCs that appear to work for their respective systems; unfortunately, there has not been a careful comparison of these various iden –
tification strategies. Notably, markers used to identify normal mammary stem cells (MaSCs) are the same as those used to iden –
tify CSCs.
22-26 It is likely that there will be significant overlap
between the markers but ultimately some will be more useful than others. In this section, we will review various methods that have been reported to effectively identify and isolate both normal and cancer stem cells.
Cd44/lin. While the existence of MaSCs was first shown
in the late 1990s by Kordon and Smith,
27 and later conclusively
proven by mammary gland development from single cells,23,24
breast CSCs were not clearly characterized until 2003.26 Clarke
and colleagues isolated a subset of cancer cells identified as CD44
high/CD24low/lin- that were able to generate tumors when
as few as 200 cells were implanted in immune-compromised mouse recipients. In contrast, cancer cells that did not display this marker set were unable to generate tumors with even 20,000 still cost prohibitive. To overcome this obstacle, investigators nar –
rowed down a 50-gene signature that can effectively differentiate the molecular subtypes using quantitative real time PCR (qRT-PCR). This 50-gene signature, termed PAM50, has been shown to be an effective replacement for full microarray analysis with an ability to classify tumors into one of the intrinsic subtypes.
19
Furthermore, it was demonstrated that a model using the PAM50 gene set for molecular classification had a significantly improved ability to predict risk of relapse as compared to a model utiliz –
ing only clinical variables (tumor size, node status and histologic grade) when tested on ER
+/node-negative patients.19 However, it
is important to mention that utilizing both clinical variables and molecular subtyping resulted in significantly better predictive value than either one alone. In light of the tremendous variability in response to therapy, it is perhaps most notable that using the molecular subtypes generated a model with 94% sensitivity and 97% negative predictive value for predicting pathological com –
plete response.
19
Thus, it is reasonable to expect that the application of molecu –
lar subtyping in clinical practice will provide useful information regarding patient-specific prognosis, risk of relapse and prob –
ability for pathological complete response. A major benefit of improved risk stratification will be the identification of patients for whom the benefits of neoadjuvant therapy outweigh the risks. Alternatively, patients with increased risk of relapse may benefit from a more aggressive treatment strategy or increased surveil –
lance. It is important to note that the PAM50 is not the only multi-gene predictor for breast cancer; there are many others that are useful for cancer classification, grading, prognosis and response to therapy (reviewed in ref. 20).
Functional Classification of Breast Cancer
Subtypes—Mammary Stem Cells
and Cancer Stem Cells
While identification of the molecular subtypes of breast cancers represents an area of significant advance in our understanding Figure 2. Molecular classification of breast cancer. This classification is based on the intrinsic molecular subtypes of breast cancer identified by micro –
array analysis of patient tumor specimens.14-16

958 Cancer Biology & Therapy volume 10 issue 10
In support of this, it was shown that the gene signature correlated
with prognosis in several other cancers including medulloblas –
toma, lung cancer and prostate cancer.
alde Fluor (aldh 1). The appeal of using cell surface
markers for the identification of CSCs is the ability to isolate via –
ble CSCs via flow cytometry. Recently, investigators have identi –
fied an intracellular enzyme aldehyde dehydrogenase 1 (ALDH1) as a marker of both normal and cancer stem cells.
22 A cell perme –
ant fluorescent substrate allows quantification of ALDH activity in live cells, facilitating the use of this marker to identify and isolate CSCs. A fluorescent substrate-based “ALDEFLUOR” assay (Stem Cell Technologies) has been shown to effectively iso –
late adult tissue stem cells from a variety of tissue types includ –
ing the hematopoietic system,
29,30 central nervous system31 and
mammary gland.22 The initial usefulness of ALDH1 in identify –
ing stem cells across tissue types is consistent with its suspected role in the early differentiation of stem cells.
32 Notably, ALDH1 cells.26 Notably, tumors generated with CD44high/CD24low/lin-
cells were able to recapitulate the histopathology of the initial tumor demonstrating the ability of these cells to regenerate the full range of tumor heterogeneity. In order to demonstrate the self-renewal ability of these cells, the authors showed that the cells retained the ability to generate tumors after serial passages.
26
These markers have now been used by several groups to isolate
CSCs from both primary tumors and established breast cancer cell lines. While this has been tremendously useful as a tool for laboratory research, its potential for clinical use relies on its ability to predict patient prognosis or response to therapy. To investigate this, an invasiveness gene signature was generated by comparing gene expression profiles from CD44
high/CD24low cancer and nor –
mal breast epithelium. This gene signature was shown to corre –
late with both overall and metastasis free survival of breast cancer patients.
28 This implies that patient prognosis may be correlated
with the level of CSCs (and hence the invasiveness gene signature). Figure 3. Functional classification of breast cancer. This scheme will classify breast cancer based on the tumor initiating cells. Currently there are two
proposed hypothesis: (A) either the heterogeneity seen in breast cancer arises from distinct mammary stem/progenitor cells at various levels within
the mammary stem cell hierarchy or, (B) breast cancer heterogeneity is the result of a single mammary stem/progenitor cell being transformed by vari –
ous oncogenes which give rise to various types of cancer.

www.landesbioscience.com Cancer Biology & Therapy 959
These studies showed that a single cell was able to reconstitute a
full mammary gland in vivo.23,24 Although the initial experiments
involving these markers were done in a mouse model system, they have successfully been utilized for isolation of MaSCs from human tissue.
39 More importantly, elevated levels of CD49f in breast can –
cer tissues correlate with poor prognosis and reduced survival. When α6-integrin levels were used in conjunction with clinical
risk factors (e.g., histologic grade, steroid receptor expression) the prognostic value was significantly better than either one alone.
40
Issues and Considerations
Failures with the one size fits all approaches to cancer therapy will no doubt be replaced by more personalized cancer therapy in the future. Understanding histological and molecular characteristics of a tumor will therefore be necessary as tools to individualize such therapy. Above, we reviewed the various approaches that can be taken to classify breast cancer, namely histological, molecular and functional. The current clinical classification system relies heavily on the histological aspects of the primary lesion; while this system has some prognostic value, it lacks a molecular basis. Through advanced technologies and tools, we are now beginning to achieve a more comprehensive understanding of breast cancer, which has led to the identification of five intrinsic subtypes of breast cancer. This represents a molecular classification/approach to breast cancer that has tremendous potential for therapeutic response and prognostic predictions. Finally, the functional clas –
sification of breast cancer is likely to use one or more of the CSC markers to quantify the percentage of CSCs in a patient’s tumor. Initial studies have clearly associated CSCs with poor clinical outcome and CSCs will likely be the focus of the next generation of targeted therapies.
Importantly, we emphasize that at present there is not a clear
basis to adopt one molecular marker or parameter over another; rather parts of several marker sets will likely need to be retained and incorporated into a more comprehensive system that will be most useful to clinicians in providing the most predictive clas –
sification of disease for the benefit of the patients. It has con –
sistently been shown that combination of clinical variables with either molecular or functional biomarkers yields a system that is more robust and capable than any one system alone. This type of approach, coupled with the rapid progress that is being made in the field of breast cancer research is likely to produce better diag –
nostic and prognostic biomarkers to improve patient outcomes.
acknowledgements
Work in our laboratories is supported by the NIH grant CA96844 and Department of Defense grant W81XWH-07-1-0351 to V.B.; the NIH grants CA87986, CA105489, CA99163 and CA116552 to H.B.; and the NCI Core Support Grant to UNMC-Eppley Cancer Center.activity has also been used to identify CSCs,
22,33 implying some
retention of normal stem cell signaling in transformed CSCs.
The ALDEFLUOR assay was originally designed to isolate
adult hematopoietic stem cells, but Ginstier et al. have recently demonstrated its ability to isolate both normal mammary stem cells and breast CSCs.
22 Although there hasn’t been a compre –
hensive comparison between the full spectrum of stem cell mark –
ers, Ginster and colleagues did compare the ALDH1 marker with CD44
high/CD24low/lin- marker set; they report approxi –
mately 20% overlap between the two markers and demonstrate that cells displaying both markers carry enhanced tumorigenic potential.
22 Moreover, they show that cells within the CD44high/
CD24low/lin- population that are able to generate tumors are also
ALDEFLUOR-positive. This suggests that CD44high/CD24low/
lin- markers are able to enrich for breast CSCs but there is likely
considerable heterogeneity within that population.
Again, the ability of a marker to predict clinical outcome is
a critical factor in determining its clinical utility. ALDH1 has been shown to correlate with the overall survival of patients by several research groups. One study using two independent sets of breast tumors with 481 collective samples found that ALDH1-positive tumors showed strong association with poor clinical outcome and high histological grade. Notably, high tumor grade has recently been associated with a higher content of CSCs.
34
Furthermore, the basal subtype of breast tumors, which are typi –
cally triple-negative and carry a poorer prognosis, contain high numbers of CSCs.
35 Another study utilizing four series of tumor
samples found a significant correlation between ALDH1 expres –
sion and basal-like breast tumors that were mostly triple-negative (ER
-/PR-/ErbB2-), which is consistent with other published data
for association with CD44+/CD24- marker set.36 More recently,
Neumeister et al. using a multiplexed flow-based assay, showed that the coexpression of ALDH1, CD44 and cytokeratins was significantly correlated with poor outcome.
37
In addition to an association with patient outcome, ALDH1-
positive tumors have also been associated with low pathologic complete response to sequential paclitaxel and epirubicin-based chemotherapy. Interestingly, the CD44/CD24 analysis was done in conjunction but did not show any significant differences in pathologic complete response rates.
38 Furthermore, it was shown
that the proportion of ALDH1-positive cells increased signifi –
cantly after neoadjuvant therapy, indicating inherent chemo –
therapy resistance in the ALDH1-positive CSCs. These studies are useful in establishing the clinical significance of ALDH1 as a biomarker for prognosis and therapy response/resistance.
Cd49f/α6-integrin and C d29/β1-integrin. In addition to
the use of CD44/CD24, several other cell surface markers have been used to isolate prospective mammary stem cells. In 2006, two groups isolated mouse mammary stem cells by first excluding cells of endothelial and hematopoietic origin and then selecting for cells based on their expression of CD24, CD29 or CD49f.
23,24

960 Cancer Biology & Therapy volume 10 issue 10
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