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Published online 9 August 2012
The relationship between components of tumour inflammatory cell infiltrate and
clinicopathological factors and survival in patients with primary operable invasive
ductal breast cancer
Background:
The importance of the components of host local inflammatory response in determining outcome in primary
operable ductal invasive breast cancer is not clear. The aim of this study was to examine the relationship
between components of the tumour inflammatory cell infiltrate and standard clinicopathological factors
including hormone status (oestrogen receptor (ER), progesterone receptor (PR) and human epidermal growth
factor receptor (HER)-2), Ki-67 and survival in patients with primary operable invasive ductal breast cancer.
Methods:
Tumour inflammatory cell infiltrate, hormone status (ER, PR and HER-2), Ki-67 and standard
clinicopathological factors were determined using routine pathological and immuno-histochemical techniques in
468 patients.
Results:
The large majority (94%) of ductal tumours had evidence of inflammatory cell infiltrate. The general
inflammatory cell infiltrate was positively associated with high grade (P<0.001), the absence of ER (P<0.001),
the absence of PR (P<0.01), the presence of vascular invasion (P<0.05) and high lymphocytic infiltrate, plasma
cell infiltrate, other inflammatory cell infiltrate and macrophage infiltrate (all P<0.001). The median follow-up
of the survivors was 165 months. During this period, 93 patients died of their cancer. On univariate analysis,
stratified for ER status, tumour size (P<0.01), lymph node involvement (P<0.001), tumour plasma cell infiltrate
(P<0.001), other inflammatory cell infiltrate (P<0.05) and treatment (P<0.05) were associated with poorer
cancer-specific survival whereas lymphocyte infiltrate (P<0.001) was associated with improved cancer-specific
survival. On multivariate analysis, stratified for ER status, lymph node involvement (P<0.05) was
independently associated with poorer cancer-specific survival whereas increased tumour lymphocyte infiltrate
(P<0.001) was independently associated with improved cancer-specific survival.
Conclusion:
The results of this study show that, using routine histology, the general inflammatory cell infiltrate was a
common feature and was positively associated with high grade, the absence of ER, the absence of PR, the
presence of vascular invasion and high-grade infiltration of lymphocytes, plasma cells, other inflammatory cells
and macrophages. Also, that within a mature cohort of patients, a high lymphocytic infiltrate was associated
with improved survival, independent of clinicopathological characteristics including ER status, in primary
operable ductal invasive breast cancer. These results rationalise previous work and provide a sound basis for
future studies in this important area of breast cancer research.
EMCC News: Breast cancer tumour make-up changes through the course of disease;
regular biopsies needed to ensure correct treatment in patients who relapse
26.09.11
Category: EMCC 2011
New research has found that breast cancer tumours change their hormonal status
throughout the course of disease, whereas the decision about the most effective
treatment for the patient is usually only based on one biopsy of the primary
tumour.
European Multidisciplinary Cancer Congress 2011, Stockholm, Sweden: For some patients, biopsy verifications
of any relapse will be very important because it may completely change their clinical management. Dr. Linda
Lindstrm, from the Karolinska Institutet Department of Oncology-Pathology, Solna, Stockholm, said that her
groups research is the first sizeable study to look at changes in tumours in multiple relapses in breast cancer
patients.
Our study demonstrates tumour instability in clinically used markers throughout tumour progression. We saw,
for example, that one in three breast cancer patients alter oestrogen (ER) or progesterone (PR) hormone receptor
status, and 15% of patients change human epidermal growth factor receptor 2, or HER2, status during the
course of disease, she said.
ER and PR receptor status tests show whether one or both of these hormones is helping to grow the cancer.
Cancer that is hormone positive can be treated by hormone-suppressing drugs, whereas hormone negative
cancers may respond to other types of treatment. Hormone negative patients are normally tested for HER2. If
this test is positive, treatment such as Herceptin will usually be given.
The researchers studied breast cancer patients in the Stockholm healthcare region who had a recurrence of the
disease between January 1, 1997 and December 31, 2007. Information on ER status in several relapses from the
same individual was assessed in 119 patients: 33.6% of patients had changes in tumour status between the
different sites of relapse (local, loco-regional and metastases) whereas 36.1% of the patients were stable ER
positive, and 30.3% were stable ER negative. Sixteen percent of patients changed from ER positive to negative
during the course of their disease, 12.6% changed from negative to positive, and 5% altered back and forth
throughout tumour progression.
In the PR group, 30.2% of patients altered their hormone receptor status, with the majority changing from
positive to negative. Until now we thought that these predictive markers remained stable during the course of
the cancer. But it is now apparent that these breast tumours markers, which are used to decide the best treatment
for the patient, change as the tumour progresses and this significantly affects the way patients respond to
particular therapies. This has important implications for the future management of the disease, says Dr.
Lindstrm.
The researchers now intend to carry out a prospective study in which they will follow a group of breast cancer
patients and examine the standard clinical markers throughout their tumour progression. With cancer
treatments becoming more and more efficacious and targeted to specific groups, it is particularly important that
the correct treatment is given throughout the disease, says Dr. Lindstrm. An additional advantage of carrying
out regular biopsies would be that they could detect other primary cancers, or benign lesions, which could spare
patients inappropriate or unnecessary therapies.
Dr. Lindstrm and colleagues think that their findings could possibly be applied to cancers other than breast.
We believe that tumour instability may be due to many different factors, for instance the choice of therapy and
other host (patient) characteristics, and that some inherent tumour behaviour may well be shared by different
tumour types. This is a promising area of research with important implications for patient management, she
concluded.
This finding is of major clinical importance because it shows that many cancer patients who relapse do not
receive optimal treatment for their disease. While the price of regular biopsies may seem high for both patients
and healthcare systems, in the long run they may avoid inappropriate and costly treatments and, even more
importantly, may be the basis for selecting more effective treatments for individual patients, said Professor
Michael Baumann, ECCO President.
ESMO spokesman Professor Fabrice Andr, from the Institut Gustave-Roussy, Villejuif, France, said: In this
study of a large series of patients whose cancer recurred, the investigators have shown molecular changes in the
tumours of more than one third. This further underlines the importance of taking regular biopsies in patients
who relapse so that they can be sure of getting the most appropriate treatment, and of running trials looking at
the relationship between the profiles of metastatic lesions and new agents specifically targeted at them.
Modern Pathology (2011) 24, 201208; doi:10.1038/modpathol.2010.191; published online 5 November 2010
Increased insulin-like growth factor-1 receptor mRNA expression predicts poor survival in
immunophenotypes of early breast carcinoma
Gloria Peir1, Encarna Adrover2, Laura Snchez-Tejada1, Enrique Lerma3, Mara Planelles4, Jos Snchez-
Pay5, Francisco I Aranda4, Daniel Giner1 and Francisco J Gutirrez-Avi1
The biology of breast carcinoma shows a great variation, reflected by the recent classification of phenotypes
based on DNA microarrays or immunohistochemistry. The aim of this study was to determine the prevalence of
insulin-like growth factor-1 receptor (IGF1R) in breast carcinoma subtypes and the impact on the outcome. We
studied 197 consecutive breast carcinoma patients in stage III treated conservatively. Phenotypes were
assessed on the basis of the expressions of ER/PR, HER2, Ki67, p53, Bcl2, CK5/6 and EGFR. Moreover,
IGF1R expression (-subunit and -phosphorylated/active form) was evaluated by immunohistochemistry,
IGF1R mRNA levels by quantitative RT-PCR and IGF1R mutations by direct DNA sequencing. Overall, 40%
(78/197) of tumors were luminal A, 24% (48/197) luminal B, 19% (37/197) HER2-positive and 17% (34/197)
basal/triple-negative. Luminal A tumors were predominantly of low grade, without necrosis, presenting in older
patients as a 2-cm unilateral mass (all P0.046). -IGF1R overexpression was observed more frequently in
luminal A (49%) cases, followed by luminal B (20%), HER2-positive area under the curve (22%) and
basal/triple-negative cases (9%) (P=0.01) with similar results for mRNA levels (53, 24, 13 and 10%,
respectively) (P=0.038), but without differences for mutations (P=NS). High IGF1R mRNA correlated with
poor patient survival among subtypes (P=0.004) (KaplanMeier; log-rank test). For overall survival, only
histological grade and IGF1R mRNA emerged as significant predictors (P0.034; Cox regression). Increased
IGF1R mRNA implies poorer patient prognosis among the different subtypes, and that may be associated with
the lack of responsiveness to tamoxifen in cases with a positive hormone receptor status. Our results highlight
the biological and clinical relevance of IGF1R in early breast carcinoma subtypes, and provide knowledge to
assist in treatment decision.
Modern Pathology (2011) 24, 209231; doi:10.1038/modpathol.2010.205; published online 12 November 2010
-Catenin pathway activation in breast cancer is associated with triple-negative phenotype
but not with CTNNB1 mutation
Felipe C Geyer1,4, Magali Lacroix-Triki1,2,4, Kay Savage1, Monica Arnedos3, Maryou B Lambros1,
Alan MacKay1, Rachael Natrajan1 and Jorge S Reis-Filho1
Aberrant -catenin expression as determined by assessment of its subcellular localization constitutes a surrogate
marker of Wnt signalling pathway activation and has been reported in a subset of breast cancers. The
association of -catenin/Wnt pathway activation with clinical outcome and the mechanisms leading to its
activation in breast cancers still remain a matter of controversy. The aims of this study were to address the
distribution of -catenin expression in invasive breast cancers, the correlations between -catenin expression
and clinicopathological features and survival of breast cancer patients, and to determine whether aberrant -
catenin expression is driven by CTNNB1 (-catenin encoding gene) activating mutations.
Immunohistochemistry was performed on a tissue microarray containing 245 invasive breast carcinomas from
uniformly treated patients, using two anti--catenin monoclonal antibodies. Selected samples were subjected to
CTNNB1 exon 3 mutation analysis by direct gene sequencing. A good correlation between the two -catenin
antibodies was observed (Spearman's r >0.62, P<0.001). Respectively, 31 and 11% of the cases displayed
lack/reduction of -catenin membranous expression and nuclear accumulation. Complete lack of -catenin
expression was significantly associated with invasive lobular carcinoma histological type. Subgroup analysis of
non-lobular cancers or non-lobular grade 3 carcinomas revealed that lack/reduction of -catenin membranous
expression and/or nuclear accumulation were significantly associated with oestrogen receptor negativity,
absence of HER2 gene amplification and overexpression, lack/reduction of E-cadherin expression and tumours
of triple-negative and basal-like phenotype. Univariate survival analysis revealed a significant association
between -catenin nuclear expression and shorter metastasis-free and overall survival in the whole cohort;
however, -catenin nuclear expression was not an independent predictor of outcome in multivariate analysis. No
CTNNB1 mutations were identified in the 28 selected breast carcinomas analysed. In conclusion, -catenin/Wnt
pathway activation is preferentially found in triple-negative/basal-like breast carcinomas, is associated with
poor clinical outcome and is unlikely to be driven by CTNNB1 mutations in breast cancer.
Review
Cell Research (2011) 21: 245257. doi:10.1038/cr.2011.11; published online 18 January 2011
From milk to malignancy: the role of mammary stem cells in development, pregnancy and
breast cancer
Benjamin Tiede1 and Yibin Kang1,2
Abstract
Adult stem cells of the mammary gland (MaSCs) are a highly dynamic population of cells that are responsible
for the generation of the gland during puberty and its expansion during pregnancy. In recent years significant
advances have been made in understanding how these cells are regulated during these developmentally
important processes both in humans and in mice. Understanding how MaSCs are regulated is becoming a
particularly important area of research, given that they may be particularly susceptible targets for transformation
in breast cancer. Here, we summarize the identification of MaSCs, how they are regulated and the evidence for
their serving as the origins of breast cancer. In particular, we focus on how changes in MaSC populations may
explain both the increased risk of developing aggressive ER/PR() breast cancer shortly after pregnancy and the
long-term decreased risk of developing ER/PR(+) tumors.
Biology of the mammary gland
The mammary gland is composed of epithelial, adipose and other stromal cells, which work in concert for the
primary goal of producing milk during nursing. In the female mouse, five rudimentary pairs of mammary
epithelial placodes begin to form from the ectoderm at E10.5 and grow until E18, at which point growth is
relatively restricted until puberty 1. This makes the mammary gland relatively unique among most tissues and
organs in that the majority of its patterning occurs in adulthood. Once gland expansion resumes during puberty,
the epithelium forms into a branching, bilayered ductal structure, consisting of an outer myoepithelial layer of
cells, which contract to help excrete milk and an inner luminal cell layer. This inner layer is subdivided into
ductal luminal cells, which line the inside of the ducts, and alveolar luminal cells, which secrete milk during
lactation (Figure 1A). In the mouse, mammary gland growth during puberty is led by the invasion of club-like
structures at the end of the ducts known as terminal end buds (TEBs), which invade into the empty adipose
tissue, dubbed the mammary fat pad. TEBs consist of an outer layer of cap cells, which eventually form
myoepithelial cells, and an inner layer of body cells, which become the luminal cell compartment (Figure
1B). The TEBs lead the growth of the gland until they reach the end of the fat pad, at which point they
disappear 2. The rest of the space in post-pubertal mammary gland is taken up by adipose tissue, along with a
mixture of blood vessels, immune cells and fibroblasts 3.
Figure 1.
Cellular structures of the mammary gland. (A) The mature mammary duct features an outer layer of
myoepithelial cells (red) surrounding an inner layer of luminal epithelial cells (blue). It is thought that
mammary stem cells (green) reside in a basal position between these two populations and give rise to progenitor
cells (teal) and both lineages of fully differentiated cells. (B) The developing mouse mammary gland invades
through the empty fat pad led by the terminal end bud (TEB). Stem-like cap cells (orange) lead invasion in the
direction of the black arrow and eventually give rise to myoepithelial cells. Many of the inner body cells
(purple) undergo apoptosis as the gland grows, with some of the progeny of inner body cells forming the
luminal cells that line the ducts of the glands.
Full figure and legend (76K)
After the extensive ductal elongation during puberty, the mammary gland undergoes minor growth and
involution during the stages of the estrus cycle until pregnancy, at which point the gland is massively
remodeled. During pregnancy, branches extend off the side of mammary ducts and proliferate to form
lobuloalveolar buds or bunches which secrete milk. During late pregnancy and into lactation, the mammary
epithelium fills the majority of the mammary fat pad. Alveolar cells secrete milk into the lumens, which is
forced through the ducts by the contractile force of myoepithelial cells. After pups are weaned, the mammary
epithelial cells undergo well-choreographed apoptosis, resulting in the involution of the gland to the point where
the parous gland returns to a structural state similar to the virgin gland. Upon the induction of further
pregnancies, these coordinated growth and involution stages repeat in a similar fashion.
The gland architecture and remodeling that occurs in the human is highly similar to what is observed in the
mouse mammary gland. However, there are some differences with respect to the gland structure. In humans the
main lobular structure observed is known as the terminal ductal lobular unit (TDLU), which exists in several
morphologically different forms throughout development and pregnancy. In the virgin gland, the relatively
undifferentiated TDLUs are termed Lob1-type, which have been described as equivalent to TEBs 4. As the
Lob1-type TDLUs begin to develop and differentiate, they form Lob2-type TDLUs, which have more ductal
structures per lobule compared to Lob1 structures. During pregnancy, the formation of even more ductules
results in the conversion of Lob2 into Lob3, which eventually form secretory acinar structures (Lob4). After
pregnancy, these TDLUs regress back in number, although in the resting parous gland the majority of TDLUs
remain as Lob2 type, whereas in virgins Lob1 are the most predominant type 4, 5, 6. In mature nulliparous
women, Lob1 structures remain the most prevalent, with a moderate population of Lob2 structures but no Lob3
or Lob4 structures. After menopause, all women show a predominance for Lob1 structures, regardless of
whether they have had children 4, 5, 6.
Because of the well-choreographed cycles of growth, remodeling and involution, researchers suspected for
many years about the existence of adult stem cells within the mammary gland. These cells would theoretically
be able to differentiate into the multiple cells of both the developing and pregnant gland, and self-renew despite
the massive apoptosis post weaning to drive the growth of subsequent pregnancies. More dubiously, properties
of mammary stem cells (MaSCs) could render them as vulnerable targets of tumorigenesis. The identity and
characteristics of both the human and mouse MaSCs have been characterized in recent years, while their
potential role during breast cancer formation is beginning to be elucidated.
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Identification of MaSCs
The two hallmark properties of any stem cell population are the ability to differentiate into multiple cell lineages
and the ability to self-renew to produce more stem cells 7. While most adult cells are unable to divide, existing
in a stage of terminal differentiation, tissue-specific adult stem cells retain the ability to divide and produce the
multiple cell types within the organ from which they are derived. In the mouse mammary gland, the first clues
for the existence of an adult stem cell of the mammary gland came from the work of DeOme et al. in the 1950s
when it was shown that small pieces of mammary epithelium, when transplanted into recipient fat pads cleared
of their endogenous epithelium, could expand and differentiate into a fully functional reconstituted gland 8.
Cells from nearly any location within the mammary gland, or during any developmental stage, could repopulate
a mammary gland 9. Subsequent experiments in both humans and mice demonstrated that this reconstitution
ability was due to the activity of a single cell. Tsai et al. suggested clonal expansion was responsible for human
mammary gland growth based on X-chromosome inactivation patterns 10, while Kordon and Smith
demonstrated through retroviral tagging that mouse mammary glands were the progeny of a single cell 11. Based
on this evidence, a number of experimental approaches were undertaken to identify and purify MaSCs based on
their biological or morphological properties.
One strategy to isolate MaSCs relies on a feature believed to be (although not universally accepted as) a key
mechanism of DNA replication during stem cell division. As certain adult stem cells divide, they preferentially
retain one of their DNA strands throughout multiple divisions in order to protect against the formation of
deleterious mutations that occur during DNA replication 12, 13. By performing pulse-chase experiments with
DNA labels, Smith et al. showed there was a population of cells within the mammary gland which retained their
DNA label through asymmetric segregation of DNA strands. These cells were still actively dividing and
featured stem cell characteristics 14. Roughly 30-40% of the cells that retained their DNA label also expressed
receptors for the reproductive hormones estrogen and progesterone 15. As an early alternative approach to label
retention, the heterogeneity of morphological characteristics of mammary epithelial cells was exploited to try to
enrich for cells with stem cell characteristics. Pale cells with low cellular complexity (i.e. few cytoplasmic
organelles) were shown to express the properties of MaSCs in differentiating conditions 9.
A major limitation of both the morphological and the label retention methods is that they do not lend themselves
to the easy isolation of large numbers of relatively pure MaSC populations for use in in vitro or in vivo assays.
As such, these methods did not definitively show that a single label-retaining cell or pale cell could reconstitute
a fully functional gland in vivo, which is the gold standard for stem cell assays. A better approach to isolate
putative MaSCs involved the simple isolation of different cell populations based on the expression of surface
marker proteins from dissociated mammary gland preparations using fluorescence-activated cell sorting
(FACS). An initial marker that showed some promise was stem cell antigen-1 or Sca1. Sca1+ cells were shown
to be a subpopulation of the label-retaining epithelial cells, and when isolated they showed a degree of in vivo
reconstitution ability. However, subsequent studies would identify markers which could enrich for MaSCs to a
much higher degree of purity, and MaSCs identified by other methods have been shown to be Sca1low/ 7, 16.
In 2006, mouse MaSCs were identified based on the expression of CD24 (heat-stable antigen) and high
expression of either CD29 (1-integrin) or CD49f (6-integrin) 7, 17. A single Lin CD24+CD29hi/CD49fhi cell
was able to reconstitute an entire mammary gland in vivo. The CD29 protein is not just a surrogate marker for
MaSCs but is actually functionally important, as CD29 ablation in the basal compartment reduced MaSC
activity 18. Multi-lineage differentiation of progenitors into luminal and myoepithelial cells was confirmed via
histological analysis and a variety of in vitro differentiation assays. The second hallmark of stem cells, self-
renewal, was confirmed via the observation of clonal gland outgrowth during serial gland reconstitution
experiments. With respect to previous markers of stemness, Sca1 did not further enrich for the MaSCs, but the
newly isolated MaSCs did seem to retain their DNA label 7. Based on expression profiling and histological
staining, the remaining non-stem cell fraction of the Lin CD24+CD29hi populations represents
basal/myoepithelial cells. Downstream progenitor and mature luminal cells are primarily observed in the
CD24+CD29lo fraction. A specific luminal progenitor subpopulation of the LinCD24+CD29lo population has
been identified based on strong expression of CD61 19. While the lineage of cells that differentiate to form the
mammary gland has not been as well characterized as systems such as the colon, a more detailed description of
the hierarchy of cells within the mammary gland can be found in the recent review by Visvader and Smith 20.
MaSCs are important for the two main growth phases of the mammary gland: the ductal elongation during
pubertal expansion and the lobuloalveolar expansion during pregnancy. However, it is unknown whether the
same population of cells with a high degree of plasticity can perform either of these functions depending on the
local hormonal and growth cues, or if MaSCs begin to differentiate early and are programmed to perform only
one of the two functions. There is evidence in other adult stem cell systems for the existence of two functionally
distinct stem cell populations within one tissue 21. Several lines of evidence support the assertion that this may
be the case in the mouse mammary gland as well. Based on label retention studies, it was discovered that
putative MaSCs existed in both basal and luminal locations 15. With the subsequent identification of better
surface marker profiles to efficiently purify MaSCs, stronger evidence emerged in parallel for the existence of
distinct MaSC populations. In the MaSC fraction based on CD24 and CD49f staining, many cells expressed the
basal maker K14. However, other cells expressed the luminal marker K18. It did not seem though that cells
expressed both of these markers, suggesting that these cells might reside in distinct locations 17. A luciferase-
based transgenic mouse model for MaSC activity did reveal luciferase-expressing cells in both basal and
myoepithelial locations 22. Notably, when the MaSC marker CD29 is deleted from the basal compartment of the
mammary gland, the mammary epithelial cells can no longer reconstitute a new mammary gland, but they can
form alveoli late in pregnancy 18, suggesting a distinct MaSC population. Similar results were also obtained
when the Wnt receptor LPR5 was deleted 23. A recent study showed that using a GFP reporter driven by the s-
SHIP promoter, GFP+ replicating active MaSCs can be identified in cap cells in puberty and basal alveolar
bud cells in pregnancy, but not in adult virgin animals, or in mammary tissues during lactation or involution
stages 16. Future characterizations will help to better understand whether or not distinct MaSC populations exist,
and how they are controlled by their local micro-environmental cues.
With respect to the human mammary gland, identification of authentic MaSCs is a greater challenge because of
the difficulty in obtaining normal tissue samples and the lack of an ideal in vivo reconstitution system.
Nevertheless, various attempts have been made to characterize human MaSCs both in vitro and in vivo. By
following similar methods of growing primary neural cells in non-adherent conditions which resulted in the
formation of neural stem cell-enriched neurospheres, mammary stem/progenitor cells could be enriched by
forming mammospheres 24. This method was further refined (for both human and mouse cells) by staining the
mammary epithelial cells with the lipophilic dye PKH26 and selecting for cells that were slow dividing and
retained this label during mammosphere growth. These cells were shown to have MaSC function in humanized
mouse mammary glands in vivo 25. An alternative isolation method was shown later by sorting cells based on
the surface maker profile of LinCD49+EpCam/lo or CD10+ and suspending these cells with irradiated human
fibroblasts in a collagen gel and then implanting them under the kidney capsule of estrogen/progesterone-treated
mice 26. It was subsequently shown that this same population of cells could differentiate into mammary gland
structures in mouse mammary glands when transplanted with supporting fibroblasts 27. Expression profiling of
human and mouse MaSCs-enriched populations has shown a significant degree of conservation in gene
expression across species 28, providing validity to using the more readily accessible mouse model. Based on
these enrichment methods for both human and mouse MaSCs, subsequent experiments have begun to elucidate
the mechanisms by which MaSCs are controlled through various signaling pathways.
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Signaling pathways important for maintaining MaSCs
A number of pathways that have been shown to play important roles in other adult stem cell systems also
function in regulating MaSCs. For instance, in the Wnt pathway the receptor LRP5 can enrich for MaSCs on its
own (although to a lesser degree than CD24 and CD29/CD49f) and is functionally required for maintaining
stem cell populations 23. Overexpression of Wnt1 using the mammary-specific MMTV promoter resulted in a
6.4-fold increase in the number of MaSCs 7. Furthermore, Wnt ligands can be used to maintain MaSCs in
culture, and when the Wnt pathway is stimulated in MaSCs they can outcompete untreated MaSCs in
reconstitution assays 29. Additionally, the Notch pathway has also been implicated in regulating MaSC fates.
Multiple reports have shown that Notch pathway ligands are expressed in the MaSC compartment, while the
Notch receptors are expressed in the downstream progenitor/luminal compartment 25, 30. Ablation of Notch
signaling through a Cbf-1 knockdown led to an expansion of MaSC activity, while forced constitutively active
Notch signaling reduced MaSC activity 30. As MaSCs divide in non-adherent growth conditions in vitro, the
Notch antagonist Numb is asymmetrically localized into only one of the daughter cells. However, in MaSCs
taken from a p53/ mouse, Numb is ubiquitously localized in both daughter cells. Not surprisingly, there is an
expansion of the MaSC population in p53/ mice 31, which seems to be the result of altered asymmetric DNA
segregation and cell division rather than the anti-apoptotic activity of p53 32. Intriguingly though, inhibition of
the Notch pathway in p53/ mammary epithelial cells reduced the MaSC activity 32, suggesting that further
research is needed to better understand the role of Notch in MaSC growth and differentiation. Finally, the p53-
related protein p63, a transcription factor known to be important for stem cell function and epithelial stasis in
other systems 33, 34, is also important for MaSCs.
p63 has previously been shown to be important for maintaining the replicative potential of basally located stem
cells in the epidermis, rather than functioning in lineage commitment or differentiation programs 34, 35. p63 is
expressed in primarily two different isoforms, N-p63 and TA-p63. The N-p63 isoform is expressed in MaSCs
of the human 27 and mouse 36, 37, where its expression can be induced by Wnt signaling 23 and promotes the
expression of self-renewal genes. The TA-p63 isoform is expressed in luminal progenitor populations and
promotes the expression of hedgehog pathway components necessary for progenitor cell function 37. Recently,
an interesting connection between p63 and Notch signaling has emerged. As mentioned, it had been shown that
MaSCs are Notch signal-sending cells, expressing the ligands on their surface, while mammary progenitors are
Notch signal-receiving cells 30. This pattern of expression is opposite to that of the basally expressed p63.
Through the use of a Notch reporter transgenic mouse it was shown that Notch and p63 were distinctly
segregated from one another 38. Intriguingly, these genes seem to be functionally antagonistic to one another.
N-p63 expression is necessary and sufficient for maintaining cells in the basal lineage, but when the activated
intracellular intermediate of Notch1 (NICD) is overexpressed, it lowers the expression of N-p63 in cells
differentiating into the luminal lineage 38. Furthermore, in Notch signaling-deficient RBP-J knockout mice, N-
p63 is aberrantly expressed in luminal cells 39, which may explain why MaSCs are known to be expanded in
Notch-deficient mice 30.
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Mammary epithelial cell dynamics during development and pregnancy
The ductal growth observed during puberty and the alveolar expansion that takes place during pregnancy are the
two main periods of extensive mammary epithelial proliferation. Recently, evidence has suggested that these
changes are driven by the coordinated division and differentiation of mammary stem/progenitor cell
populations. MaSCs localized in the cap region of the TEB are responsible for the growth during the ductal
elongation phase in puberty 7, 17 (Figure 1B). New data have also supported the notion that MaSCs are important
for the growth observed during pregnancy. Intriguingly, an apoptosis-resistant population of cells known as
parity-induced mammary epithelial cells (PI-MECs), which arise during pregnancy after activation of the whey
acidic protein (WAP) promoter, show stem cell characteristics 40, 41. PI-MECs are a heterogeneous population,
but generally express the MaSC markers CD24 and CD49f, and when transplanted can reconstitute mammary
glands.
Further analysis based on both non-invasive in vivo tracking 22 or FACS analysis combined with gland
reconstitution assays 22, 42 has shown a significant expansion both in total number and in percentage of MaSCs
during pregnancy. The peak of this expansion seems to occur mid- to late-pregnancy at the end of alveolar
proliferation and the onset of differentiation to begin milk production. Although s-SHIP promoter-driven GFP
reporter marks active MaSCs in both the puberty and pregnancy stages 16, it is unknown whether pregnancy-
associated MaSCs come from the same stem cell population important for pubertal growth, or if a distinct
MaSC population exists or is responsible specifically for growth during pregnancy. While the start of milk
production corresponds with the drop in MaSC numbers, sustained milk production continues to affect the rate
of MaSC apoptosis, as when mothers do not nurse their young MaSC numbers drop quicker than those who do
nurse their young 22. Eventually, well after weaning of pups, a statistically significant reduction in the
percentage of MaSCs has been observed in young but not old mice 22, 43, 44. During pregnancy, MaSC expansion
comes at a cost of self-renewal capability. This was demonstrated by Asselin-Labat et al. 42, who showed a
defect in secondary gland reconstitution from donor cells taken from pregnant mice. Thus, it is not surprising
that by tracking MaSCs in individual mice using an in vivo model, a smaller expansion of MaSCs has been
observed in second pregnancies 22.
Downstream of MaSCs, the progenitor/luminal population also expands extensively and then involutes, but with
an understandably delayed onset compared to the MaSCs 22. By refining this population based on CD61
staining, it has been shown that the CD61+ progenitors do not rise in total number until later in pregnancy
during lactation 19, 42, which suggests that the first increase in the combined CD24+CD29lo population observed
before pups are born 22 is likely to be CD61 cells. Since CD61+ progenitors do not express hormone receptors
but CD61 cells do 19, the initial rise of CD61 cells is likely a direct effect of the hormone signaling, but the
second wave of CD61+ proliferation is likely the result of division of MaSCs into progenitors.
Given that mammary stem/progenitor cell dynamics change extensively during puberty and pregnancy, it is not
surprising that the reproductive hormones estrogen, progesterone and prolactin play important roles in this
regulation. In general, estrogen signaling is important for ductal elongation during puberty 45 and also during the
early stages of pregnancy 40; progesterone is also important for the initial side branching observed early in
pregnancy 46 and prolactin is important for differentiation late in pregnancy 47. In each of these cases, indirect
paracrine signaling was shown to be important for the expansion of the mammary gland, which is not surprising
since MaSCs and progenitors have been shown to not express receptors for these hormones 19, 36 and hormone
receptor-positive cells generally do not readily proliferate 48. A number of regulators and downstream targets of
these hormones have been identified that control the growth and differentiation of MaSCs and progenitors. For
instance, the prolactin target STAT5A is necessary and sufficient for the formation of CD61+ luminal
progenitors in virgin mice 49, 50 while targets ELF5 and GATA-3 are important during differentiation of CD61+
luminal progenitors during pregnancy 19, 51, 52. With respect to MaSCs, the progesterone receptor regulator
C/EBP is needed to maintain the MaSC pool in virgin mice, and is also important during pregnancy 53, 54.
Another pathway that seems to be particularly important for MaSC expansion during pregnancy is the
RANK/NF-B pathway. It has been known that the pregnancy-associated hormones progesterone, prolactin and
PTHrP increase the expression of the RANK ligand (RANKL) 55 while knockout of RANK, RANKL or
inhibition of the downstream kinase IKK results in a defect in lobuloalveolar expansion and milk secretion
during pregnancy 55, 56. This defect is largely due to impaired activity of the cell cycle-promoting factor Cyclin
D1 56. Recent evidence has begun to suggest this effect is specific to MaSCs. Through expression profiling, it
was shown that RANK expression is elevated in MaSCs during pregnancy or in response to hormone treatment,
while RANKL is expressed in luminal cells 42. This leads to the activation of the stem cell factor ID2 in MaSCs.
When pregnancy hormones were removed via ovariectomy, a loss of RANK signaling was observed, as well as
a significant loss in the number of functional MaSCs 42. Notably, RANK inhibition directly limited the colony-
forming ability of sorted MaSC populations, suggesting a functional role 42.
Given this evidence for the role of RANK/NF-B signaling in promoting alveolar cell proliferation through
activation of MaSCs, it is not surprising that this pathway is often associated with breast cancer formation 57.
Furthermore, this could help explain the propensity for breast cancer metastasis to bone, since this pathway
constitutes an important part of the vicious cycle of osteolytic bone metastasis 58. Unfortunately, this pathway
is not alone among MaSC-regulatory factors that are also important drivers of tumorigenesis. As such, mounting
evidence implicates a role for MaSC growth during breast cancer formation.
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MaSCs and breast cancer
Because of their relatively long life span and ability to undergo self-renewing divisions, adult stem cells have
been suggested as ideal candidates for the initial transforming events that drive cancer formation. While this has
been demonstrated for leukemia 59, evidence in solid tumors has not been as clear cut. It is important to
remember that breast cancer is a heterogeneous set of diseases, each with their own etiology, course of
progression and outcome. Nevertheless, it is possible that MaSCs could serve as the cell of origin for distinct
classes of breast cancer. Based on clustering analysis of gene expression data from a cohort of human breast
cancers, five major tumor types were identified 60, 61. The basal type of tumors is frequently (but not always)
triple negative for the expression of ER, PR and ErbB2/Neu. Clinically this subtype of tumors is of great
interest because it is associated with a poor patient prognosis 60, 61. MaSCs also lack the expression of these
receptors 36, suggesting that they may be the tumor-initiating cell. Additionally, a number of previously
identified regulators expressed by MaSCs such as Notch ligands, p63 and components of the Wnt pathway are
known to be involved in basal-type tumors and are associated with poor outcomes 62, 63, 64. Furthermore, markers
of the epithelial-mesenchymal transition (EMT) are preferentially expressed in the basal and claudin-low
subtypes of breast cancer 65, 66, 67. Notably, multiple facets of the EMT process are thought to play an important
role in expansion and invasion of the MaSC-rich TEBs during development 68, and it was also shown that forced
EMT produces mammary cells with stem cell and tumorigenic characteristics 69. More substantial investigation
has further implicated that transformation of mammary stem or progenitor cells might drive tumorigenesis for
distinct tumor types and ultimately effect patient prognosis. Much of this evidence fits into the paradigm of
tumor-initiating cancer stem cells (CSCs).
It is believed that tumors, much like mature tissues and organs, are comprised of a hierarchy of cells which
contain differing degrees of replicative and differentiation capacity. CSCs are the subpopulation of a
heterogeneous tumor, which when separated and transplanted can self-renew and differentiate into tumors
matching the initial degree of heterogeneity. The remaining tumor cells are devoid of this property. By
determining the identity of the original cell population that was transformed to form the CSC population,
researchers will be able to identify key steps early in the formation of various cancers, and potentially reveal
novel therapeutic targets which would allow for analyzing the root cause of tumor formation.
Multiple features of stem/progenitor cells make them likely candidates as the origin of CSCs. Stem/progenitor
cells generally are very long lived compared to committed cell populations, which would provide a greater
window for them to accumulate the multiple genetic mutations necessary for transformation. Furthermore, the
self-renewal capability that stem/progenitor cells possess predisposes them with the replicative potential needed
for overt tumor formation. Although stem cells often have mechanisms to protect against DNA damage (such as
asymmetric segregation of DNA strands), loss of these protective mechanisms is often a hallmark of tumor
formation. Several lines of evidence suggest that CSCs exist in tumors of other tissues/organs and may be
derived from stem/progenitor cells (reviewed in 70, 71, 72); however, only evidence concerning breast CSCs will be
discussed here.
In a landmark paper in 2003, Al Hajj et al. showed that a subpopulation of human tumor cells with the surface
marker expression profile of CD44+CD24/lo could form heterogeneous tumors in serial transplantation assays 73.
However, given the multiple mechanisms that can drive breast cancer initiation, there are likely to be multiple
different CSC populations with potentially different cells of origin depending on the oncogenic event 7, 74.
Mouse models provide a simple way to evaluate different transformation events. In p53/ mice, there are
elevated numbers of MaSCs 31, and the MaSC marker profile CD24+CD29hi can enrich for breast CSCs 75.
However, in the MMTV-Wnt1 and p53+/ models, the mammary progenitor marker CD61 enriched for breast
CSCs 74. Intriguingly, the CD61+ cells in the MMTV-Wnt1 tumors showed some mammary repopulating ability
normally reserved for stem cell populations, suggesting that a re-adoption or improper expression of stem cell
characteristics could be an early event during tumorigenesis in this model. This could also explain the expansion
of MaSCs observed in pre-neoplastic tissue in this strain 7. Breast CSCs were also identified in MMTV-Wnt1
mice by the expression of CD24 and Thy1 (Thy1 has been used as a stem cell marker in other tissues and
organs). When profiled, these cells produced a signature that was similar to published MaSC gene expression
signatures 76. Other global gene expression profiling analyses suggested that the mouse MaSC signature
correlated with not only the MMTV-Wnt1 tumors, but also p53/ tumors 28. The CD61+ luminal progenitor
population most closely resembled MMTV-Neu and MMTV-PyMT tumors, while committed luminal cells
resembled MMTV-Myc tumors 28.
As for human breast tumors, a variety of evidence suggests that MaSCs or progenitors may serve as the targets
for transformation. Because of the difficulty in performing transplantation experiments of pre-neoplastic tissue
with human tissues, gene expression profiling has often been used as a surrogate to show common expression
patterns between normal mammary epithelial cells and particular tumor types, suggesting a possible cell of
origin. For instance, by globally profiling the miRNAs that are differentially expressed between human breast
CSCs and non-CSCs, a set of miRNAs were identified that are also differentially regulated between normal
mammary gland stem/progenitor cells and committed cells 77. In particular, mir-200 was expressed in the non-
stem cell populations which suppressed Bmi1 expression that is needed to promote self-renewal and block
differentiation 77. Alternatively, a normal human MaSC signature was shown to be up-regulated in either
undifferentiated or basal-like tumors 25. However, using a different MaSC isolation method, it was shown that
MaSCs showed overlap in gene expression profiles with claudin-low/normal breast-like tumors, while the
luminal progenitors correlated highly with the basal cancer and committed luminal cells looked most like the
luminal subtype tumors 27. In the same study, Lim et al. were able to perform transplantation experiments from
pre-neoplastic tissue similar to the experiments performed with mouse models by taking tissues from breast
cancer-susceptible Brca1 mutation carriers. It was suggested that in Brca1 carriers luminal progenitor cells
served as the targets for tumorigenesis 27. Both BRCA1 and BRCA2 have previously been implicated in the
normal differentiation process of the mammary gland 78, 79. Interestingly, in the Lim et al. study, the Brca1
mutation carriers had lower MaSC numbers but higher numbers of luminal progenitors in normal glands.
However, the progenitors from Brca1 mutation carriers showed higher colony-forming ability than non-carriers
(and even showed higher colony-forming ability than MaSCs in the absence of the growth supplement B27),
suggesting an altered mammary hierarchy resulting from either stem or progenitor cell dysfunction.
In addition to these experimental studies, a variety of long-term observational studies have revealed associations
suggesting that MaSCs or progenitor cells play an important role during tumorigenesis. For instance, many
years after the massive radiation exposure of the nuclear bombs dropped on Hiroshima and Nagasaki, the cohort
of women who showed the highest incidence of subsequent breast cancer development were those entering
puberty at the time of the bombs, when MaSC activity is expected to be elevated 80, 81. Additionally, breast
cancer was the most frequent cancer in women who received chest irradiation during adolescence for treatment
of Hodgkin's disease 82, 83. Some of the strongest observational evidence, though connecting mammary
stem/progenitor cells with breast cancer, focuses on the role of pregnancy in affecting breast cancer
susceptibilities.
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Pregnancy, breast cancer and MaSCs
For many years, epidemiological studies have demonstrated that an early, full-term pregnancy at young age is
the only feature known to lower the lifetime risk of breast cancer without respect to race or ethnicity 4, 84, 85, 86, 87.
Women who are younger than 24 years old at the time of their first full-term pregnancy are protected against
developing breast cancer much later in life, while women over the age of 35 are ultimately at an increased risk
88, 89
. However, immediately following the first full-term birth, women are at an increased risk of developing
breast cancer 90, which in younger women lasts around 10 years, but in older women lasts longer 91. Pregnancy
generally protects against the development of ER/PR(+) tumors, while the tumors that form shortly after
pregnancy usually do not express these hormone receptors 84, 92, 93 and are generally more aggressive. Because
pregnancy results in such a strong, universal long-term protective effect against developing breast cancer,
understanding the mechanism behind this effect could provide ideal targets to mimic this natural protective
mechanism. Fortunately, this phenomenon is also observed in a variety of rodent models of chemically induced
mammary tumors 4, 94, opening avenues to pursue experimental channels to understand the mechanism.
In general, there are four somewhat overlapping explanations to account for the protective effect (reviewed in
84
), all of which involve the impact on MaSCs to varying degrees (and are not mutually exclusive). Pregnancy
could alter the levels of circulating hormones within the mammary gland, alter the hormone responsiveness of
the cells within the gland itself, promote a more differentiated, growth-refractory state of the gland as a whole,
or alter the number of MaSCs which could serve as the targets for transformation. Here, we will focus primarily
on the role of MaSCs as the targets for transformation and how this may lead to an increased risk of developing
aggressive ER/PR() tumors shortly after pregnancy and a decreased risk of developing ER/PR(+) tumors long
after weaning (Figure 2).
Figure 2.
Potential roles of MaSCs during pregnancy and tumorigenesis. (A) In the normal virgin mammary gland,
ER/PR() MaSCs (green) exist in a relative balance with ER/PR(+) mature cells (blue, red). (B) During
pregnancy, the number of MaSCs expands due to symmetric self-renewing divisions. This large pool of MaSCs
may serve as the direct targets of transformation for ER/PR() tumors, leading to a short-term increased risk of
breast cancer. (C) After weaning, through differentiation and involution, the number of MaSCs is lower than the
resting gland and the remaining MaSCs have lower self-renewal ability. This lowers the overall risk for
developing ER/PR(+) breast cancers, which may result either from transformation of MaSCs, which as they
differentiate require additional oncogenic mutations to compensate for the reduced self-renewal ability before
forming tumors, or from the direct transformation of ER/PR(+) committed cells.
Full figure and legend (38K)
Two recent reports have shown in mouse models that a significant expansion of MaSCs occurs during
pregnancy, peaking before the time that pups are born 22, 42. A number of features of tumors that form shortly
after pregnancy suggest that direct transformation and expansion of MaSCs may drive the formation of these
tumors. As mentioned above, pregnancy-associated tumors are ER/PR(), similar to MaSCs themselves.
Furthermore, tumors forming shortly after pregnancy in humans show elevated Her2 levels while showing
decreased expression of the cell cycle inhibitor p27 and decreased levels of the p27 inducer BRCA1 95.
Numerous pieces of evidence suggest that these may be critical effectors in transforming MaSCs during
pregnancy.
With respect to Her2/Neu, tumors from MMTV-Neu transgenic mice are ER() and are composed of a fairly
homogeneous population of luminal cells. Intriguingly, pregnancy-associated tumors which arise in the MMTV-
Neu strain are derived from the stem cell-containing PI-MEC population 96. Within the MMTV-Neu tumors,
there are cells that could divide and produce cells which simultaneously expressed both luminal and
myoepithelial cytokeratin markers, even though the tumors themselves were primarily luminal 97. This
suggested that the tumors retained some degree of stem/progenitor cell activity, which is understandable given
that MMTV-Neu tumors show a loss of p53 which is linked to deregulated stem cell asymmetric division 31.
One critical mediator of Neu-driven pregnancy-associated breast cancer that affects MaSCs is Cyclin D1.
Cyclin D1 knockout mice show defects in lobuloalveolar development and lactation during pregnancy and are
refractory to tumor development when crossed with the MMTV-Neu strain 98, 99, 100. The inactive Cyclin D1
K112E mutant shows a specific defect in PI-MEC cell self-renewal and differentiation during pregnancy 101,
suggesting lower numbers of cells susceptible to tumor development. Not surprisingly, this strain also inhibits
MMTV-Neu-driven tumor formation 101. While mammary glands from these mice showed lower mammary
reconstitution frequency, it appeared there was a specific defect in colony-forming progenitor cells, rather than
MaSCs themselves 101. Because the transgenic MMTV promoter for these experiments is pregnancy hormone
responsive, the data observed with the MMTV-Neu mice in relation to pregnancy should be interpreted
cautiously until further confirmation is shown through more advanced knockout, conditional overexpression or
lentiviral overexpression experiments.
In addition to Her2/Neu, another factor important for MaSCs that may help explain the increased risk of
developing aggressive tumors shortly after pregnancy is BRCA1. BRCA1 tumors are typically ER/PR negative
basal-type and tumor-initiating CSCs from BRCA1 mutation carriers can be isolated using markers of normal
MaSCs 102. BRCA1 is expressed in MaSC-enriched TEBs of the developing mouse mammary gland and its
expression is elevated during pregnancy by combined stimulation of estrogen and progesterone 78. Functionally,
BRCA1 knockdown led to an increased amount of secondary and tertiary mammosphere formation and led to
improper differentiation in vivo 103. Not surprisingly, BRCA1 levels are reduced by 33% and p27 levels are
reduced by 89% in pregnancy-associated breast cancers 95. Taken together, these observations suggest that
changes in p53, Her2/Neu and BRCA1 expression in the expanded MaSC compartment may contribute to the
increased risk of developing tumors shortly after giving birth (Figure 2B). These and other factors may also play
a role in changing MaSC levels to protect against developing breast cancer long after pregnancy.
As mentioned above, many years after giving birth at young age, women develop ER/PR(+) breast cancers at a
decreased rate compared to women who have not undergone a full-term pregnancy. Multiple studies have
confirmed that in young mice, pregnancy ultimately decreases the number of MaSCs in the mammary gland 22,
44
. Again, if MaSCs are the targets for transformation, this could help explain the lower breast cancer rates in
women having undergone a full-term pregnancy. Notably, this effect dissipates with increased age at the time of
first pregnancy 88, 104, and in the mouse model pregnancy in older mice did not result in decreased MaSC
numbers 43. However, unlike the tumors that form shortly after pregnancy, the protective effect long after
pregnancy is against ER/PR(+) tumors, suggesting that in such cases mutations that occur in MaSCs may not
directly lead to transformation, but could lead to tumor formation upon further oncogenic challenges in
downstream progenitors (Figure 2C). This may partly be explained by the observation of altered self-renewal in
MaSC populations after one full-term pregnancy and smaller expansion in successive pregnancies 22, 42. This
suggests that the cells that remain after involution of the gland are not as growth-competent and thus it may take
more oncogenic insults to form tumors. A similar change in the characteristics of MaSCs after pregnancy has
been proposed in human mammary glands, where pregnancy is known to result in the progression of Lob1
TDLUs into more differentiated Lob2, Lob3 and Lob4 structures. Russo et al. suggested that pregnancy results
in a conversion of Stem Cells 1 found in the undifferentiated Lob1 structures into more differentiated Stem
Cells 2 (roughly equivalent to mouse PI-MECs) found in more differentiated structures 4 which are resistant to
tumor formation. When tumors do arise in the more differentiated TDLUs, they are less malignant.
If lowering the number of MaSCs is crucial for the pregnancy-associated breast cancer protection, then
deregulation of the apoptotic machinery in MaSCs during involution or promotion of stem cell growth should
correlate with a loss of the protective effect. (It is already known that improper apoptosis after hormone level
decrease during the estrus cycle promotes tumorigenesis in progenitor cells in a transgenic model 105.) Not
surprisingly, p53 activity, which is important for the normal involution process after pregnancy, is required for
parity-dependent breast cancer protection, while loss of p53 leads to an increase in MaSC activity through
increased symmetric cell divisions 31, 106.
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Perspectives
With the discovery of new markers to better identify and track MaSCs, clear insights have been gained
regarding the role of MaSCs during developmentally important processes such as puberty and pregnancy.
However, our understanding of the role of MaSCs in tumorigenesis remains opaque compared to other systems.
In order to better elucidate this role, future studies will greatly benefit from attempts to further refine the makers
used to isolate MaSC populations to better purify MaSC fractions (and possibly distinct fractions important for
gland elongation during puberty versus gland expansion during pregnancy). Additionally, it will be important to
be able to better track MaSC populations in vivo to directly test their susceptibility to transformation in
particular forms of breast cancer. By doing so, researchers will be able to ascertain how regulating MaSC
numbers during pregnancy ultimately impacts parity-associated breast cancer risk and also begin to determine
which of the key signaling pathways important for MaSCs in development also play a role during
tumorigenesis. These insights will be particularly important in coming years given that women are electing to
have children later in life, which results in a decreased or mitigated protective effect. Therefore, research to
understand the mechanisms behind the protective effect will hopefully lead to the ability to better predict breast
cancer susceptibility in high-risk patient groups during pregnancy and potentially even strategies to induce or
enhance this protective effect when needed.
Next Section
Abstract
OBJECTIVE. We determined the pattern of spread of metastatic lobular carcinoma in the chest, abdomen, and
pelvis on CT.
MATERIALS AND METHODS. We identified 57 women (age range, 30-79 years; mean age, 57 years) with
metastatic lobular carcinoma of the breast who underwent CT of the chest, abdomen, or pelvis between 1995
and 1998. Then two experienced oncology radiologists retrospectively reviewed 78 CT examinations of those
patients to identify sites of metastatic disease and to identify complications caused by metastases.
RESULTS. Metastases were identified in bone in 46 patients (81%), lymph nodes in 27 patients (47%), lung in
19 patients (33%), liver in 18 patients (32%), peritoneum in 17 patients (30%), colon in 15 patients (26%),
pleura in 13 patients (23%), adnexa in 12 patients (21%), stomach in nine patients (16%), retroperitoneum in
nine patients (16%), and small bowel in six patients (11%). Eighteen patients (32%) had gastrointestinal tract
involvement that manifested as bowel wall thickening. Hydronephrosis was present in six patients (11%).
CONCLUSION. Although lobular carcinoma metastasized to common metastatic sites of infiltrating ductal
carcinoma, lobular carcinoma frequently metastasized to unusual sites, including the gastrointestinal tract,
peritoneum, and adnexa. Gastrointestinal tract involvement was as frequent as liver involvement, appearing as
bowel wall thickening on CT. Hydronephrosis was a complication of metastatic lobular carcinoma.
Previous SectionNext Section
Introduction
Breast carcinoma is the most common malignancy in women [1], with an estimated 180,200 new cases
diagnosed annually in the United States [2]. Infiltrating ductal carcinoma, the most common histologic subtype
of breast carcinoma, accounts for approximately 90% of all invasive breast carcinoma. Invasive lobular
carcinoma differs from infiltrating ductal carcinoma in its histology and mammographic appearance on the basis
of the tendency of malignant cells to surround the mammary ducts and lobules in single file, often creating a
targetoid appearance, without forming glandular aggregates [3]. Although lobular carcinoma accounts for only
10-14% of all breast carcinoma [3], given the high incidence of breast carcinoma, lobular carcinoma affects a
large number of women; its incidence is greater than that of invasive cervical carcinoma and approximately two
thirds that of ovarian cancer [2].
The radiologist plays an integral role in examining patients with lobular carcinoma, not only in the detection of
the primary lesion but also in the identification of metastatic disease and evaluation of treatment response. With
the advent of multiple chemotherapeutic and hormonal agents for the treatment of patients with metastatic
breast carcinoma, recognition of metastatic disease and its potential complications has become increasingly
important. The CT appearances of metastatic breast carcinoma have been described [4, 5]. However, most
studies do not distinguish between patients with lobular carcinoma and those with infiltrating ductal carcinoma
and likely reflect patients with infiltrating ductal carcinoma. Because lobular carcinoma has a distinct histology
and certain mammographic features that differ from those of infiltrating ductal carcinoma, it is possible that
lobular carcinoma also has a different pattern of metastatic spread. Although there have been a few imaging
case reports about a small number of patients with metastatic lobular carcinoma [6,7,8], to our knowledge no
large series exists describing the relative prevalence of different metastatic sites of lobular carcinoma as seen on
CT. Because knowledge of the pattern of disease spread is essential for accurate image interpretation, we
undertook this study to determine the distribution of metastatic lobular carcinoma in the chest, abdomen, and
pelvis as seen on CT.
Discussion
Lobular carcinoma is a distinct subtype of breast carcinoma that differs from infiltrating ductal carcinoma in its
histologic appearance. The radiologist often plays an integral role in examining patients with lobular carcinoma
to detect metastatic disease and to assess treatment response. The pattern of spread of metastatic lobular
carcinoma to the central nervous system differs from that of infiltrating ductal carcinoma. Whereas infiltrating
ductal carcinoma is more likely to produce cerebral masses, lobular carcinoma commonly produces
leptomeningeal carcinomatosis [11]. We undertook this study to determine the distribution of metastatic lobular
carcinoma in the chest, abdomen, and pelvis as seen on CT.
In our series, bone was the most common site of metastatic lobular carcinoma. Although lobular carcinoma also
metastasized to lymph nodes, lung, and liver (common metastatic sites for patients with infiltrating ductal
carcinoma), it frequently metastasized to the gastrointestinal tract, peritoneum, and adnexa. Gastrointestinal
tract involvement was as frequent as liver involvement, appearing as bowel wall thickening on CT. The
predilection of lobular carcinoma to metastasize to the gastrointestinal tract, peritoneum, and adnexa seen in this
study is in accordance with findings of large autopsy and clinical studies [12,13,14] (Table 3). Interestingly,
Harris et al. [12] reported a higher rate of spread of metastatic lobular carcinoma to the peritoneum or
retroperitoneum than to bone at autopsy. Lamovec and Bracko [13] reported an equal rate of spread of
metastases to peritoneum and bone. Our observation that lobular carcinoma metastasized most frequently to
bone likely reflects, in part, the greater sensitivity of CT in the depiction of osseous metastases than of
peritoneal and gastrointestinal tract lesions. In our study, CT was performed with routine scanning protocols and
was not tailored for the specific evaluation of the gastrointestinal tract. It is possible that if effervescent tablets
had been administered to maximize gastric distention and if rectal contrast material had been administered, then
even more bowel or peritoneal lesions may have been revealed.
TABLE 3 Distribution of Metastatic Sites of Lobular Carcinoma (LC) and Infiltrating Ductal Carcinoma
(IDC) Reported at Autopsy and in Clinical Findings
Although we did not directly compare the distribution of metastatic lobular carcinoma with that of metastatic
infiltrating ductal carcinoma, the autopsy studies that do directly compare these carcinomas [12, 13] report a
significantly higher prevalence of spread of metastatic disease to the gastrointestinal tract, peritoneum and
retroperitoneum, and ovaries in patients with metastatic lobular carcinoma compared with patients with
metastatic infiltrating ductal carcinoma (Table 3). In a clinical study of 2605 patients with invasive breast
carcinoma (lobular carcinoma [n = 359] and infiltrating ductal carcinoma [n = 2246]), Borst and Ingold [14]
reported a significantly higher prevalence of spread of metastatic lobular carcinoma to the gastrointestinal tract,
peritoneum and retroperitoneum, and gynecologic organs than metastatic ductal carcinoma. Metastases in that
study were assessed by histologic, radiologic, and physical examination. Taal et al. [15] retrospectively
identified 17 patients with metastatic breast carcinoma to the colon or rectum over a 15-year period. Fifteen of
these patients (88%) had lobular carcinoma and only one (6%) had infiltrating ductal carcinoma [15]. In another
study, Taal et al. [16] identified 27 patients with metastatic breast carcinoma to the stomach during the same
time: 20 (74%) had lobular carcinoma and only four (15%) had infiltrating ductal carcinoma. Patients in both of
these studies were examined with single or double contrast-enhanced fluoroscopy and endoscopy. The reason
for these differences in the metastatic patterns of lobular carcinoma and infiltrating ductal carcinoma is
unknown. It has been suggested that loss of expression of the cell-cell adhesion molecule E-cadherin in
infiltrating lobular carcinoma may contribute to these differences [17].
In our series, the most common appearance of metastatic lobular carcinoma to the gastrointestinal tract was
tumor infiltration along the bowel wall with mural thickening (Figs. 1,2,3). No patient in our study had an
isolated mural mass on CT. Although it is sometimes difficult to distinguish serosal implants and true mural
involvement on imaging, histopathologic examination confirmed metastatic infiltration within the bowel wall in
six patients. Meyers [18] described three different radiologic appearances of hematogenous metastases to the
bowel: an intramural mass (as seen in patients with metastatic melanoma), a mesenteric mass with secondary
invasion of the bowel wall (as sometimes seen in patients with lung carcinoma), and linitis plastica (bowel wall
thickening and rigidity). Linitis plastica, the most common appearance of metastatic breast cancer to the bowel,
is usually described in the stomach.
Initial fluoroscopic descriptions of linitis plastica caused by metastatic breast carcinoma do not report the
histologic subtype of the primary tumor in most patients [19, 20]. In a subsequent study, Cormier et al. [21]
retrospectively identified 31 patients with linitis plastica caused by metastatic breast cancer. All the patients had
metastatic lobular carcinoma, and no patient had metastatic infiltrating ductal carcinoma. Harris et al. [12]
reported at autopsy that lobular carcinoma metastasized to the stomach in a diffuse spreading process and
with a linitis plastica-like appearance in the most severe cases. The cases of gastric metastases caused by
infiltrating ductal carcinoma adopted a nodular configuration. The mural thickening and rigidity described
with metastatic breast carcinoma is caused by the dense infiltrate of tumor cells along the bowel wall [18, 19].
This parallels the appearance of lobular carcinoma in the breast. One distinguishing feature of primary lobular
carcinoma is the infiltration of malignant cells in single file, often producing an asymmetric density on
mammography with no dominant mass [22].
On imaging, linitis plastica caused by metastatic breast cancer may be indistinguishable from primary scirrhous
carcinoma of the stomach [20, 23, 24]. Signet cells have been described in both lobular carcinoma and primary
gastric carcinoma. A gastric biopsy that reveals adenocarcinoma with signet cell features caused by metastatic
lobular carcinoma may be mistaken for a primary gastric carcinoma. The treatment for these two diseases varies
greatly. Whereas gastrectomy may be a treatment option for patients with primary gastric carcinoma, systemic
chemotherapy or hormonal therapy would be instituted for patients with metastatic lobular carcinoma.
Therefore, it is particularly important to be aware of a history of primary lobular carcinoma, because further
evaluation with immunohistochemistry may aid in distinguishing these two entities [25].
Patients with genitourinary [26] or gastrointestinal [27, 28] metastases may develop significant complications
that warrant intervention. Grabstald and Kaufman [29] reported a series of 24 patients with hydronephrosis from
periureteral breast metastases. Asch et al. [27] reported a series of 12 patients with metastatic breast carcinoma
to the gastrointestinal tract with complications requiring surgical treatment, including bowel obstruction,
gastrointestinal hemorrhage, and pneumoperitoneum caused by a perforated metastasis. Six patients (11%) in
our study had hydronephrosis caused by metastatic infiltration of the retroperitoneum. Both large- and small-
bowel resections were performed as treatment of intestinal obstruction caused by metastatic lobular carcinoma
(which developed subsequent to the CT examinations). One patient in our study underwent appendectomy for
symptoms of appendicitis, and histopathologic examination revealed tumor infiltration in the appendiceal wall.
Our institution is a tertiary referral center that treats a large number of patients with breast cancer who present at
all disease stages; therefore, we have the opportunity to examine many patients with metastatic lobular
carcinoma. It is possible that our data reflect a selection bias for patients with more advanced disease. However,
the distribution of metastases in our investigation is in direct accordance with the largest clinical study
published thus far evaluating sites of metastatic lobular carcinoma, which included 359 patients with invasive
lobular carcinoma (both with and without metastatic disease) over a 17-year period [14] (Table 3).
The limitations of our study should be addressed. Our study was retrospectively performed and reports the
various sites of distant metastatic lobular carcinoma on CT. As with any imaging study, we cannot conclude that
our results reflect the exact prevalence of metastases to different sites. However, our findings do reflect the
results of two major autopsy studies (which theoretically have the highest accuracy in assessing prevalence) [12,
13]. We did not assess for leptomeningeal carcinomatosis, a finding described with metastatic lobular carcinoma
[11], because CT images were obtained with a large field of view for the evaluation of the chest, abdomen, and
pelvis. Chest wall recurrences are usually clinically evident, and CT scans are not routinely obtained to detect
local tumor recurrence. Therefore, we reviewed CT scans for distant metastases only. Because biphasic contrast-
enhanced CT scans were not routinely obtained, we did not characterize the enhancement pattern of hepatic
metastases as hypervascular or hypovascular.
The purpose of this study was not to assess the sensitivity or specificity of imaging in the detection of metastatic
breast carcinoma, but rather to determine the distribution of metastatic lobular carcinoma. Although we do not
have pathologic proof for all sites thought to represent metastatic disease on CT, pathologic confirmation of
metastatic breast cancer was available for at least one site per patient in 44 patients (77%). In clinical practice,
once metastatic disease is documented in a patient, biopsy of additional lesions is usually not required. Strong
clinical suspicion of metastatic disease existed in an additional 13 (23%) patients on the basis of physical
examination, elevated tumor markers, and correlative imaging studies. Most metastases in this group of 13
patients were in the bone, lung, pleura, and liver. None of these 13 patients had metastases in the gastrointestinal
tract or peritoneum on CT. We purposefully included these 13 patients to avoid a selection bias for patients with
atypical metastatic sites that required biopsy. We excluded patients who had a second primary malignancy to
ensure that the metastatic lesions revealed on imaging were from lobular carcinoma. The authors recognize, as
with all imaging studies, radiologic review is not equivalent to proven metastatic disease.
We did not correlate the specific sites of metastatic disease with the time elapsed since different types of
therapy. It is possible that various types of chemotherapy, hormonal therapy, or immunosuppressive therapy
(bone marrow transplantation) may alter the pattern of metastatic disease. However, a large-scale study
evaluating the precise relationship of therapy to the onset of metastases at various sites with CT scans obtained
at regular time intervals would be required to accurately address this question.
In conclusion, although lobular carcinoma metastasized most frequently to bone, lobular carcinoma has a
propensity to metastasize to the gastrointestinal tract, peritoneum, and adnexa (relatively atypical sites for
patients with infiltrating ductal carcinoma). Gastrointestinal tract involvement was as frequent as hepatic
involvement in this series, appearing as bowel wall thickening on CT. The radiologist should be aware of the
histologic subtype of breast carcinoma when interpreting body CT for metastatic disease. Knowledge of this
pattern of disease spread will not only aid in the detection of metastatic disease and its potential complications,
but also aid in minimizing the possibility of mistaking metastatic disease for a second primary malignancy.
Clinical Study
British Journal of Cancer (2012) 107, 221223. doi:10.1038/bjc.2012.273 www.bjcancer.com
Published online 26 June 2012
Patterns of metastasis in women with metachronous contralateral breast cancer
V Vichapat1, H Garmo1,2, L Holmberg1,2, I S Fentiman3, A Tutt4, C Gillett3 and M Lchtenborg1,5
Background:
The understanding of metastatic patterns after metachronous contralateral breast cancer (CBC) may help
determine the biological nature of CBC.
Methods:
A cohort of 8478 women with breast cancer treated at Guys and St Thomas NHS Foundation Trust between
1975 and 2006 were studied. Organ-specific 5-year cumulative incidence and incidence rate ratios were
assessed for women diagnosed with unilateral breast cancer (UBC), CBC within 5 years and CBC more than 5
years of the initial diagnosis.
Results:
Women diagnosed with CBC within 5 years had a higher incidence of metastases in all organs compared with
UBC. Women with a short interval time to CBC developed metastasis more rapidly and were more likely to
develop visceral and distant cutaneous metastases compared with bone metastasis.
Conclusion:
These findings explain poor prognosis of women with early occurring CBC and suggest that some of these
CBCs are indicators of aggressive and/or systemic disease.
Slide 1.
Lisa A. Carey, MD: Hi, I'm Lisa Carey. I'm the Medical Director of the Breast Center at the University of
North Carolina. I'd like to welcome you to this Medscape Oncology CME lecture titled "The Biology of Breast
Cancer."
Slide 2.
The overall goal of this activity is to describe the various intrinsic subtypes of breast cancer and to discuss how
to select and apply appropriate treatment, particularly for patients with metastatic disease, on the basis of tumor
biology. To help us measure the educational effectiveness of this activity, please take a moment to answer the
following questions.
Slide 3.
It's important to recognize that breast cancer is not one disease but is made up of a family of diseases that are
biologically distinct from one another. The understanding of this has come from gene expression array-type
analyses, which have been done by a number of research groups and have had generally concordant findings.
For example, as seen here, unsupervised analysis -- which means examining the tumors without any knowledge
of how the cancers behaved (whether they relapsed or not or whether the patient died or not) but simply asking
about differences in biology -- gives us a great understanding of baseline differences among the different
subtypes. There are certain groups of genes that differentiate breast cancers into discrete groups, and these genes
are those you might have predicted. They include hormone receptor-related genes, HER2-related genes, a
unique group of genes called the basal-like genes, and proliferation genes. When those gene clusters are used to
segregate breast cancers, you come up with at least 5 -- although there may end up being more -- intrinsic
subtypes, including at least 2 of the luminal subtypes A and B, which make up the majority of hormone-
receptor-positive breast cancers; the basal-like; the HER2-enriched; and the claudin-low type, which is more
recently described.
Slide 4.
The first group that we should consider, because it's the most common, is the luminal, subtypes A and B. These
are characterized by high expression of the hormone-receptor-related gene cluster. The HER2 gene cluster can
either be highly expressed or not, and HER2 itself can either be positive or negative. They can have a variety of
proliferation signals, and this is the most heterogeneous group.
Slide 5.
The HER2-enriched subtype makes up about 15%-20% of tumors and is characterized by high expression of the
HER2 cluster but also low expression of the hormone-receptor-related cluster. Those tumors that are high for
both HER2 and hormone receptors fall into the luminal category. So this refers only to those that are likely to be
HER2 negative and ER negative on clinical assays. It's typically a very proliferative group.
Slide 6.
The claudin-low subtype is a very interesting one. It's much more recently described, and we have as many
questions as we have answers about it. It's also relatively uncommon. It makes up no more than 10% of the
tumors. It's one of the triple-negative breast cancers. It's also characterized histologically by often having a
lymphocytic infiltrate, and it does have expression of immune-related genes on its gene-expression array. One
of the most important features, though, is that it has very low expression of cell-cell junction proteins, which
may make it more migratory, and it has characteristics that are reminiscent of the stem cell, which is part of the
reason for a lot of research interest in this group.
Slide 7.
The basal-like makes up the majority of triple-negative breast cancers -- about 15% of tumors. It's characterized
again by low expression of the HER2 and the hormone-receptor-related gene clusters -- which is why it's triple
negative -- and high expression of that basal cluster that includes several cytokeratins, EGFR, and a number of
other genes. These are typically highly proliferative, with evidence of genomic instability; 50% of them are
p53-mutant, comprising the majority of breast cancers in BRCA1 mutation carriers. If you focus on the bottom
of the slide, from p53 mutations down, these features are generally considered to be evidence of abnormal DNA
damage repair pathways.
Slide 8.
Although these intrinsic subtypes were identified using expression profiling, which is still the best way to
identify them, it can't be applied in the clinic. What are often used as a surrogate are clinical assays, which, of
course, are far easier and accessible but a lot less accurate. There are a number of groups trying to develop
assays that can intrinsically subtype clinically accessible specimens. Thus far, the PAM50 has been developed,
although there are likely to be others that can be used in fixed tissue to give us subtyping. It's important to note
that at this point, what we have in the clinic are ER, PR, and HER2 to use as these proxies. That's important
because there is misclassification whenever you're using a surrogate for an intrinsic subtype.
Slide 9.
If you're trying to determine the biology of a basal-like breast cancer, for example, and you're using the triple-
negative surrogate, it's important to note that you will identify triple-negative breast cancers that are not basal-
like on gene expression array, which constitute about 25% of the tumors. Conversely, there are also going to be
basal-like tumors that are not triple negative. They can have expression of ER, PR, or HER2. So when we talk
about triple-negative breast cancer we're mostly, but not entirely, talking about the basal-like molecular subtype.
Slide 10.
There are certain characteristics that distinguish these subtypes on the basis of clinical features. For example,
our group and many others subsequently noted that young African American women are far more likely to have
the basal-like subtype than are either older women or white women, as is shown on this table. The basal-like
subtype comprises 39% of young African American women, whereas it makes up only about 15% of all the
other groups. This finding has been replicated in larger datasets. These tumor subtypes don't particularly differ
by stage or by nodal status to any significant degree. The basal-like subtype and the HER2-enriched subtype --
shown here as HER2 positive, ER negative -- are usually ductal and they're usually grade 3.
Slide 11.
In addition to differences in clinical characteristics, there's a very intriguing suggestion from at least 2 groups
that the risk factors may differ by subtype. What's shown here is risk factor reanalysis according to
immunostains from the Carolina Breast Cancer Study, which is a very large case-control study that oversampled
young women and African American women. It's actually quite uniquely suited to answer these questions. As
you see in this table, the traditional risk factors can vary in the extent of their effect between luminal A breast
cancers, which were identified as ER positive and HER2 negative, or the basal-like, which were identified by a
5-biomarker immunostain. You see, for example, that young age of menarche looks like it is a far stronger risk
factor for basal-like breast cancer, as is having a waist-hip ratio or centripetal obesity for basal-like subtypes
compared with luminal. In addition, the protective effect of breast-feeding, which is considered modest in
unselected breast cancers, actually looks like it's statistically significant in the basal-like subtype. Even more
interesting is the suggestion that certain risk factors actually change their direction of effect. For example,
having multiple children, which is traditionally considered a protective factor, is so for luminal A, but it's
actually a risk factor for basal-like breast cancer. Similarly, being quite young at first birth is a protective effect
in luminal A subtypes but a risk factor in basal-like. This is something that will need to be further examined in
large datasets that are designed specifically to look at each factor by subtype. But if confirmed, epidemiologists
have suggested that 68% of basal-like breast cancer in young African American women may, in fact, be able to
be prevented through lifestyle modifications like weight control and breast-feeding.
Slide 12.
There are prognostic implications of the breast cancer subtypes. Shown here are a series of Kaplan-Meier
curves using the PAM50 intrinsic subtyping method on a large dataset of fixed specimens. Looking at the
Kaplan-Meier curve labeled A, you can see that the luminal A subtype, as in all datasets, has the best prognosis.
The luminal B, which is the other hormone-receptor-related breast tumor subset, actually has a far worse
prognosis. This distinction between A and B is something that is going to be important for us in the clinic. The 2
hormone-receptor-negative subtypes, the basal-like and the HER2-enriched, carry the worst prognosis, as has
been shown in other datasets. In fact, without HER2-targeted treatment, HER2-enriched breast cancer carries
the worst prognosis. The panels B, C, and D are really designed to highlight one important concept, and that is
that intrinsic subtype is not always revealed by clinical assays. In fact, if you look at patients with ER-positive
group tumors by clinical assays as in panel B, you will see that although most of them show up as either luminal
A or B, there are a few basal-like and HER2-enriched cancers in there. Similarly, in the ER-negative subset
shown in panel C, on intrinsic subtyping, a few of them are a luminal subtype. It's the same for the HER2-
positive tumors on clinical assays. This underscores my earlier comment about the fact that clinical assays can
be used as a surrogate but not a terribly accurate one for the intrinsic subtypes.
Slide 13.
If you look at these intrinsic subtypes and compare them with the kind of genomic profiles that we've all
become very familiar with (ie, prognostic profiles), you can see that there are certain subtypes associated with a
uniformly poor prognostic profile. For example, the basal-like and HER2-enriched tumors, which we know
carry a poor prognosis, have a very poor signature on Recurrence Scores, MammaPrints, and the activated
wound-healing signature. Luminal B subtypes also typically have a poor signature. The greatest variability is
actually seen in the luminal A group, which, because it's the largest group, is the one we need the most help
with.
Slide 14.
Another interesting feature of the different subtypes of breast cancer is that they not only have different
prognoses, but they also have different patterns of relapse. There's a tendency to consider relapse as a binary
thing, but we all know that there's a great deal of difference between early and late relapse and relapse in
different sites. In fact, the subtypes differ in that respect. For example, the risk for relapse over time varies
among the subtypes (shown on the top graph). There's an early peak for relapse in the triple-negative breast
cancers that falls off after about 5 years. Conversely, the other groups, which are largely made up of luminal
breast cancers, have a relatively constant risk for recurrence. In fact, after you get to 5, 6, or 7 years out, the risk
for recurrence for patients with the luminal subtypes is actually higher than for those with triple-negative.
In addition to differences in the relapse risk over time, the pattern in terms of sites of involvement also differs
(shown in the bottom table). The likelihood of visceral involvement is far higher in a HER2-positive breast
cancer or a triple-negative breast cancer. On the other hand, the likelihood of bone involvement is far higher in
hormone-receptor-positive breast cancer. Notably, CNS involvement, which is important because it requires a
completely different approach to therapy, is higher in both triple-negative and HER2-positive tumor subtypes,
and those are groups for which targeted therapies directed at the CNS are currently in active investigation.
Slide 15.
So, what about treatment? In truth, we already treat according to subtype: Luminal subtypes receive endocrine
therapy; the HER2-enriched subtypes receive HER2-targeted therapy; the luminal B, which typically have high
Recurrence Scores, often receive combined chemoendocrine therapy; the basal-like subtypes receive
chemotherapy; and patients with HER2-negative breast cancers, when appropriate in the metastatic setting,
receive bevacizumab.
Slide 16.
Of course, this is a general challenge for the future in that we'd like to be smarter about how we give
chemotherapy -- whether we can choose the chemotherapy agent [according to subtype] -- and whether there are
new targeted agents that are relevant in certain subtypes.
Slide 17.
Let's talk about antiangiogenic therapy first. What is shown in this slide are the Kaplan-Meier curves from the
seminal bevacizumab study, ECOG 2100, in which patients with metastatic HER2-negative breast cancer
received either weekly paclitaxel alone or with bevacizumab added as first-line therapy and showed a
remarkable improvement in progression-free survival [when bevacizumab was added]. The question is which
patients derived benefit, because this is a generally unselected group of patients (outside of the HER2
negativity) and that's not the way we'd like to use our targeted treatments.
Slide 18.
Efforts to determine which patients derived the benefit are shown here in these forest plots, which suggest
essentially the same benefit regardless of hormone receptor, previous chemotherapy, age, or any other variable.
So that was not helpful. In truth, the same thing has been seen in the AVADO study [docetaxel + bevacizumab].
Slide 19.
Unfortunately the intrinsic subtypes do not help us here. There is not any clear evidence of a greater or lesser
benefit across the different intrinsic subtypes, and while many investigators have tried to develop angiogenic or
hypoxic profiles, whether these can help to clinically determine benefit of antiangiogenic strategies has not been
shown.
Slide 20.
Let's talk a little more about endocrine therapies. Luminal A breast cancers are more common. They typically
have higher expression of hormone receptors and related genes. They are typically lower in terms of
proliferation genes. They can have variable Recurrence Scores and MammaPrint results. They generally
have a better prognosis. Conversely, luminal B subtypes have lower hormone-receptor expression, although it is
still present, and higher proliferation. And they all tend to have the higher Recurrence Scores, suggesting less
hormonal sensitivity and more chemosensitivity. And they have the worst prognosis.
Slide 21.
What are the ways that we may be able to get around endocrine resistance? For any of the luminal subtypes,
endocrine therapy is going to be the keystone of treatment. The most obvious approach to trying to address
endocrine resistance would be through pharmacogenomic means. For example, efforts at tying tamoxifen
efficacy to cytochrome P450 2D6 genotype and the efficacy of converting tamoxifen to its active metabolite,
that's an area of very active research. The jury is still out about how to use pharmacogenomic approaches. There
is also a great deal of data regarding EGFR crosstalk with the hormone-receptor signaling pathways. This has
been very clearly demonstrated in the research setting. I don't think there's any question about crosstalk among
EGFR and several of the other signaling pathways, but the clinical data regarding EGFR inhibition as an adjunct
to estrogen-receptor targeted therapies have been very conflicting. At this point, this remains a research tool.
There are also FGFR pathways that appear to be relevant, particularly in certain subsets of the luminal subtypes.
PI3 kinase pathway alterations appear important in endocrine resistance, and, in fact, PI3 kinase mutations that
activate the pathway are far more common (about a third) in luminal breast cancers than in other subtypes.
Slide 22.
This is potentially a way forward. These are intriguing data suggesting that targeting this pathway
[PiK3Ca/AKT], in addition to targeting the hormone-receptor signaling pathways, may be a useful strategy.
In this study, investigators took nearly 300 postmenopausal patients with hormone-receptor-positive breast
cancer and treated them in a neoadjuvant setting. They received either the aromatase inhibitor letrozole alone or
with the mTOR inhibitor everolimus, with mTOR being late down in that PI3 kinase AKT pathway. What they
found was that the response rate was significantly increased with the addition of the mTOR inhibitor to simple
aromatase inhibition. Even more intriguing from the standpoint of "proof of principle" was that although they
saw an antiproliferative effect that was increased with the addition of everolimus (RAD001) across all groups, it
was particularly true of those patients who had activating mutations, as shown in the bar graph at the bottom of
the slide.
Slide 23.
As I mentioned, the Recurrence Score helps to differentiate luminal A vs B subtypes, and that is certainly a
clinical way forward for us.
Slide 24.
As is shown here, if you extrapolate from the high Recurrence Scores, then patients with luminal B tumors
would be much more likely to be chemosensitive and to derive benefit from adding chemotherapy to endocrine
therapy. What's shown in this slide is the percentage increase in distant relapse-free survival at 10 years from
NSABP B20 [National Surgical Adjuvant Breast and Bowel Project, Trial B-20] with the addition of MF
[methotrexate, fluorouracil] or CMF [cyclophosphamide, methotrexate, fluorouracil] to tamoxifen by
Recurrence Scores. This has also been shown with CAF [cyclophosphamide, doxorubicin, fluorouracil] in
SWOG-8814 [Southwest Oncology Group, Trial 8814]. The assumption is that if you knew a patient had a
luminal B subtype of tumor, you could extrapolate that she is likely to have a high Recurrence Score, and this
benefit would also accrue to her.
Slide 25.
What about HER2-targeted therapy? The HER2-enriched tumor subtypes typically are negative for hormone
receptors, so we are reliant on chemotherapy and HER2 targeting. Fortunately, this is an area of not only great
interest, but also where there have been quite substantial advances over the last few years. From the standpoint
of HER2 targeting, we don't know whether there's any difference between targeting a HER2-positive luminal
tumor or targeting a HER2-positive, ER-negative or HER2-enriched tumor, but the therapeutic partners
certainly are different. Endocrine partners are usually added for HER2-positive luminal tumors. You can add
either small molecules, such as lapatinib, or monoclonal antibodies, such as trastuzumab. Both have been shown
to improve outcome over aromatase inhibitors alone in the metastatic setting. Patients with HER2-enriched
tumors are treated with a chemotherapy partner or, more emergently, dual HER2-targeting with lapatinib and
trastuzumab. The way that we move forward is to even more effectively use HER2 blockade than we already
do. The landscape right now includes trastuzumab, the monoclonal antibody, and lapatinib, the small molecule.
What's shown on this cartoon are the multiple other approaches that are quite reasonable for targeting this
particular signaling pathway. Shown on the upper right-hand side are other monoclonal type approaches. There's
particular interest right now in the trastuzumab-DM1 complex, which adds a maytansine analog -- a cytotoxic
complex -- to trastuzumab, a kind of "antibody-plus" approach. Other growth factor receptors appear to be
important in crosstalk for this signaling. So, similar to what we discussed with ER signaling, it appears that
targeting other growth factor receptors may be key to improved HER2 targeting. PI3 kinase, AKT, and other
signaling pathways downstream from HER2 also may be key. There is a lot of active research in this area right
now.
What we can take home from recent clinical trials is that ongoing HER2 targeting, even after progression,
appears to be relevant and important, particularly if patients are selected for having a HER2-driven breast
cancer. There's also some suggestion that dual targeting may be better than single targeting. Lapatinib added to
trastuzumab after trastuzumab failure appears to be better than merely switching from one to another. HER2 and
ER appear to be unlike certain other signaling pathways; they appear to be very targetable by themselves, and as
single pathways, you can shut them down.
Slide 26.
I'm going to talk about chemotherapy because I think it tends to get a bad rap nowadays. We should remember
that chemotherapy is indicated in the vast majority of our patients at some point in their course of disease. It has
been shown to improve quality of life in the stage IV setting, and it is the favored partner for all targeted agents,
other than endocrine therapy. Although our focus is on rationally designed targeted treatments, in truth,
chemotherapy is not going away.
Slide 27.
I'd also argue that we tend to think of these drugs as "smart drugs" vs "dumb drugs." The dumb drugs are those
with nonspecific off-target effects, typically chemotherapy -- witness the hair loss, marrow suppression, etc. The
rationale for targeted agents is that they've become much more specific for the target, more highly efficacious,
and have fewer off-target effects on the normal tissues. The question, of course, is whether chemotherapy is
quite as dumb as we give it credit for, and can we make it smarter?
Slide 28.
We know that the intrinsic breast cancer subtypes vary substantially in their sensitivity to conventional
cytotoxics. What's shown here is the pathologic complete response to neoadjuvant anthracycline/taxane-based
chemotherapy, showing that the basal-like and HER2-enriched tumors have a far higher likelihood of pathologic
complete response rate. Of the hormone-receptor- positive subtypes, the luminal B has a much higher likelihood
of pathologic complete response rate, at about 18%, and essentially there are no pathologic complete responses
with luminal A's. This has been confirmed in other datasets, and in this setting it was done as intrinsic
subtyping.
One question about this is whether this is specific to the anthracycline/taxane-based regimen that was used.
Slide 29.
What's shown here on the next slide is a heat map of the proliferation gene set, which is one of the gene sets that
characteristically differentiates the intrinsic subtypes. If you just blow up those genes and take a look at them,
you can see that there's a suggestion that any subtype that's highly proliferative may, in fact, be generally
chemosensitive. As you can see, many of the targets of our conventional agents circled here already show up in
the proliferation gene set. This may provide a clue that there is a general chemosensitivity and not necessarily
sensitivity to particular agents.
Slide 30.
Is there a way to choose specific chemotherapy drugs? Much of this work has been based on our understanding
of BRCA1-associated breast cancer. Let me start off by reminding you that when women who carry the BRCA1
mutation develop breast cancer, they do not get a whole host of different kinds of cancer; they virtually always
get the basal-like subtype of breast cancer. It's important to note that while most cancers in BRCA1-mutation
carriers are basal-like, most basal-like breast cancers are sporadic. They don't arise in BRCA1-mutation carriers.
Slide 31.
In fact, BRCA1-associated breast cancer is uncommon: It only comprises about 5%-10% of all breast cancers.
However, there are shared characteristics of BRCA1-associated breast cancers and sporadic basal-like breast
cancers, which has been termed "BRCA-ness." BRCA-ness characteristics are listed in the box on this slide.
These include high grade, triple negativity, certain histologic characteristics, p53 mutations, EGFR expression,
certain inactivation patterns of the X chromosome, and sensitivity to DNA damage.
Slide 32.
So why would we care? Why would it matter if BRCA1-associated and sporadic basal-like breast cancers are
similar? It really comes back to the fact that the BRCA1 pathway is a key mediator of DNA damage repair,
which has implications for both chemosensitivity and PARP inhibition, which is a novel target for a class of
agents of great interest right now.
Slide 33.
What are the cytotoxic or chemotherapy implications of BRCA1 dysfunction? This is all theoretical, but a
number of groups have surmised that because BRCA1 is so important in cell-cycle arrest in the setting of DNA
damage, where it causes checkpoint induction so that the cell can repair the DNA damage, that would confer
resistance to DNA damaging agents if BRCA1 is present. On the other hand, when BRCA1 is lost, checkpoint
control is lost, the cell is unable to repair its DNA, and that would confer additional sensitivity to DNA
damaging agents, with the classic ones being platinum agents.
Slide 34.
Are there clinical data to support this? In truth, there are some data but not very many. I'm highlighting 2 of the
most important pieces of data here. As you see, there was a group of more than 100 patients with BRCA1-
associated breast cancers, studied retrospectively. These patients were treated with a variety of chemotherapy
agents, and part of them included a group of 12 patients treated on a clinical trial with cisplatin only. When the
investigators compared the BRCA1-associated tumors across the different regimens, they noted that a very high
proportion of those patients receiving single-agent cisplatin, specifically 83% of them, or 10 out of 12, had
pathologically complete responses, whereas all of the other chemotherapy regimens produced no higher than
22% pathologic complete responses. This was considered suggestive of a higher sensitivity to platinum agents
than other agents in these BRCA-associated tumors. It should be noted that this is a retrospective study and the
cisplatin trials were systematically different from the others. But these findings are intriguing.
Prospective data came from a trial of cisplatin alone, where 22% of the patients who had triple-negative tumors
had pathologic complete responses to cisplatin. This response rate is far lower in the triple-negative setting,
raising a question about whether triple negativity actually selects for BRCA-ness from this standpoint. It also
should be noted that the 2 known BRCA1 carriers in that study did have pathologic complete responses. I'd say
the clinical jury is still out on platinum sensitivity, certainly for the triple-negative subtypes.
Slide 35.
What about PARP inhibition? The use of PARP inhibitors is based on the concept of synthetic lethality that's
illustrated on this cartoon. The idea is that in the setting of DNA damage, a normal cell has several ways to
repair its DNA. What's highlighted here are the base excision repair, called BER, and the homologous
recombination of systems for DNA damage repair. Homologous recombination is a BRCA1-dependent function.
In the setting of BRCA loss, DNA damage is preferentially repaired by the BER pathway, and that does work.
Conversely, the BER pathway is PARP deficient, so when PARP is lost or inhibited, then the cell is entirely
dependent on other means, such as homologous recombination. However, if you lose both pathways, the tumor
becomes unable to repair DNA using either pathway, which tends to increase sensitivity to cytotoxicity.
Slide 36.
What about clinical proof?
There's been some proof of principle with small-molecule inhibitors of PARP, such as olaparib, that were
administered to known BRCA1 and 2 mutations carriers, where a very high response rate was seen even in a
heavily pretreated population.
Slide 37.
In terms of triple negativity, there are also some data suggesting that this may be a successful approach from the
study looking at BSI-201 added to gemcitabine/carboplatin chemotherapy in metastatic triple-negative breast
cancer. What's shown here is the Kaplan-Meier curve. To summarize these results, you see a marked
improvement in progression-free survival with the addition of the PARP inhibitor to the
gemcitabine/carboplatin, from 3.3 to 6.9 months.
Slide 38.
Even more intriguing, results of this randomized phase 2 study suggested a survival advantage with the addition
of the PARP inhibitor: Overall survival improved from just under 6 months to just over 9 months. The
registration phase 3 trial for this particular approach has been completed and we eagerly await the results.
Slide 39.
There are a number of PARP inhibitors in development, as highlighted on this table. So there may be a number
of drugs available coming down the pike.
Slide 40.
I'd like to provide some cautionary notes about targeted therapies in these different subtypes, however. For
example, EGFR, which is a known target within the basal-like gene cluster and has been shown in tissue
microarrays and cell lines of the basal-like subtype to be quite important, has been tested in 2 trials that have
already been published and one that will be reported later this year.
Slide 41.
Essentially, there was modest clinical activity seen with the addition of EGFR inhibition to chemotherapy in
patients with triple-negative breast cancers.
Slide 42.
In one of these studies, patients with accessible tissue were asked whether they would allow serial biopsies of
their target tumor. What you see here is a visual representation based on one of the patients who allowed this to
be done. The cartoon represents her EGFR pathway. You can see EGFR expressed on the top and you see all the
bubbles representing the downstream molecules in the EGFR pathway. At the very bottom are 3 lines of genes
that were determined ahead of time to represent activation of the pathway. You can see that because those
clusters are more red than green, this is considered an EGFR-activated tumor. One week later, with the addition
of EGFR inhibitor to carboplatin, all of those 3 lines representing EGFR activation were turned off (green), and
this represents EGFR inactivation. In fact, this patient was a strong clinical responder.
Slide 43.
However, as George Sledge [George Sledge, MD, current President of the American Society of Clinical
Oncology] once said, "One dumb tumor is still smarter than 10 smart oncologists." In the same case, if you look
on the left, the pretherapy tumor, you see EGFR that's expressed, and on the bottom, you see the EGFR
activation clusters. They're pretty strongly uniformly red or "on." One week later, absolutely nothing has
happened, and this tumor progressed rapidly through therapy. This was true for 16 tumors. In some, there was
no evidence of EGFR activation, so there was no chance of the drug exerting any effect. In the remainder, which
is the majority, although it looked like EGFR was activated and should have been targetable while it was
activated, the drug did nothing. Only in a minority of the tumors was the pathway activated and the drug
worked.
Slide 44.
It's important to remember the truth about targeted therapies: that it's harder to do what we're trying to do than
we think. Even if you just take EGFR as shown here, what appears to be a clean system when you're using
preclinical models becomes very messy and complex, with multiple ligands, heterodimerization, mutation of the
receptors, and all sorts of altered downstream signaling when you get to the human condition and human
tumors. Cancers typically have redundant modular pathways, which means that single agents are likely to fail in
most. We're going to have to get smarter about this, which means that we will have to embed tissue-based
studies into our clinical trials.
Slide 45.
In summary, the intrinsic subtypes reflect real biologic differences among different classes of breast cancers.
There really is a fundamental difference between hormone-receptor-positive and hormone-receptor-negative
disease. Clinicians have known it for years and researchers agree. The intrinsic subtypes are biologically
different, and the way forward involves trials in selected populations. I hope that the days of unselected breast
cancer trials are over. This may have implications for specific chemotherapy and targeted treatments. I would
argue that the future depends on the alignment of tissue-based and therapeutic trials, and I would observe that
the reason that so many of the cooperative clinical trials groups are doing neoadjuvant studies is recognition of
exactly this issue.
Slide 46.
Please take a moment to complete the activity post-test and earn CME credit. I thank you for your interest in
this Medscape Oncology CME activity.
Molecular Diagnostics
British Journal of Cancer (2010) 103, 10341039. doi:10.1038/sj.bjc.6605873 www.bjcancer.com
New relationships between breast microcalcifications and cancer
R Baker1,2, K D Rogers2, N Shepherd2,3 and N Stone1,21Biophotonics Research Unit, Gloucestershire Hospitals NHS Foundation Trust, Great Western Road, Gloucester,
GL1 3NN,UK2Cranfield Health, Cranfield University (Shrivenham Campus), Shrivenham, Swindon, Wiltshire, SN6 8LA,UK 3Department of Histopathology,
Gloucestershire department already included Hospital NHS Foundation Trust, Great Western Road, Gloucester, GL1 3NN, UKCorrespondence: Dr N Stone, E-mail:
n.stone@medical-research-centre.com
Abstract
Background:
Breast microcalcifications are key diagnostically significant radiological features for localisation of malignancy.
This study explores the hypothesis that breast calcification composition is directly related to the local tissue
pathological state.
Methods:
A total of 236 human breast calcifications from 110 patients were analysed by mid-Fouries transform infrared
(FTIR) spectroscopy from three different pathology types (112 invasive carcinoma (IC), 64 in-situ carcinomas
and 60 benign). The biochemical composition and the incorporation of carbonate into the hydroxyapatite lattice
of the microcalcifications were studied by infrared microspectroscopy. This allowed the spectrally identified
composition to be directly correlated with the histopathology grading of the surrounding tissue.
Results:
The carbonate content of breast microcalcifications was shown to significantly decrease when progressing from
benign to malignant disease. In this study, we report significant correlations (P<0.001) between
microcalcification chemical composition (carbonate content and protein matrix:mineral ratios) and distinct
pathology grades (benign, in-situ carcinoma and ICs). Furthermore, a significant correlation (P<0.001) was
observed between carbonate concentrations and carcinoma in-situ sub-grades. Using the two measures of
pathology-specific calcification composition (carbonate content and protein matrix:mineral ratios) as the inputs
to a two-metric discriminant model sensitivities of 79, 84 and 90% and specificities of 98, 82 and 96% were
achieved for benign, ductal carcinoma in situ and invasive malignancies, respectively.
Conclusions:
We present the first demonstration of a direct link between the chemical nature of microcalcifications and the
grade of the pathological breast disease. This suggests that microcalcifications have a significant association
with cancer progression, and could be used for future objective analytical classification of breast pathology. A
simple two-metric model has been demonstrated, more complex spectral analysis may yeild greater
discrimination performance. Furthermore there appears to be a sequential progression of calcification
composition.
Translational Therapeutics
British Journal of Cancer (2010) 103, 12011208. doi:10.1038/sj.bjc.6605909
Modulation of plasma complement by the initial dose of epirubicin/docetaxel
therapy in breast cancer and its predictive value
Abstract
Background:
Despite the widespread use of neoadjuvant chemotherapy in breast cancer patients, prediction of individual
response to treatment remains an unsolved clinical problem. Particularly, administration of an inefficient
chemotherapeutic regimen should be avoided. Therefore, a better understanding of the molecular mechanisms
underlying response to neoadjuvant chemotherapy is of particular clinical interest. Aim of the present study was
to test whether neoadjuvant chemotherapy with epirubicin/docetaxel induces early changes in the plasma
proteome of breast cancer patients and whether such changes correlate with response to therapy.
Methods:
Plasma samples of 25 breast cancer patients obtained before and 24h after initiation of epirubicin/docetaxel-
based neoadjuvant chemotherapy were analysed using two-dimensional differential gel electrophoresis (2D-
DIGE). Protein spots found to be differentially expressed were identified using mass spectrometry and then
correlated with the pathological response after six cycles of therapy. Markers identified in a discovery set of
patients (n=12) were confirmed in an independent validation set (n=13).
Results:
2D-DIGE revealed 33 protein spots to be differentially expressed in response to chemotherapy, including the
complement factors C1, C3 and C4, inter--trypsin inhibitor, -1-antichymotrypsin and -2-Heremans-Schmid
glycoprotein (AHSG). With respect to cytokines, only interleukin (IL)-6, IL-10 and soluble intracellular
adgesion molecule 3 (sICAM3) were minimally modulated. Moreover, two protein spots within the complement
component C3 significantly correlated with response to therapy.
Conclusion:
We have identified acute phase proteins and the complement system as part of the early host response to
epirubicin/docetaxel chemotherapy. As complement C3 cleavage correlates with the efficacy of
docetaxel/epirubicin-based chemotherapy, it has the potential as an easily accessible predictive biomarker.
As shown in an earlier study, Hes-1 is repressed by ER at the transcriptional level.[16] We therefore suggest that E2 treatment of ER+
breast cancer cells leads to inactivation of Hes-1, both directly and through the induction of Hes-6. However, the role of ER in Hes-6
regulation is not known and must be clarified in future studies. Interestingly, as shown in Figure 4, treatment of MCF-7 cells with the
selective estrogen-receptor modulator (SERM) tamoxifen caused repression of Hes-6, indicating that Hes-6 might work as a marker
for tamoxifen response in breast cancer cells. In addition, it is possible that repression of Hes-6 could be important for the breast
cancer-suppressive effects of tamoxifen.
Conclusions
Based on our findings, we propose that Hes-6 has an important role in the proliferation of breast cancer cells. Hes-6 is expressed in
low levels in normal breast tissue but is strongly induced in breast cancer tissue. As an ER-regulated gene, Hes-6 constitutes a novel
link between estrogen signaling and the Hes family of proteins, which are involved in differentiation and proliferation. Consequently,
a better knowledge of this signaling pathway could be important for the identification of endocrine-resistant tumors. Furthermore,
Hes-6 seems to be essential in ER-mediated induction of E2F-1, a critical step in the G1/S-phase transition of the cell cycle. Hes-6
emerges as a potential marker for breast cancer and might be a target for novel treatments based on the Hes signaling pathway.
Modern Pathology (2010) 23, 13571363; doi:10.1038/modpathol.2010.123; published online 25 June 2010
Histological features of medullary carcinoma and prognosis in triple-negative basal-like
carcinomas of the breast
Felicia Marginean1, Emad A Rakha1, Bernard C Ho1, Ian O Ellis1 and Andrew HS Lee1
Medullary carcinomas have a better prognosis than other grade 3 mammary carcinomas, but they typically show
basal-like biological features, which are associated with a poor prognosis. In this study we examined the
associations and prognostic relevance of medullary histological features in a series of 165 invasive carcinomas
with a basal-like phenotype: triple-negative (oestrogen receptor, progesterone receptor, HER2) and expressing
at least one basal marker (CK5/6, CK14, CK17 or EGFR). The following histological features were associated
with each other: prominent inflammation, anastomosing sheets, absence of fibrosis, absence of infiltrative
margin and absence of gland formation. Prominent inflammation and anastomosing sheets in at least 30% of the
tumour were associated with a better prognosis on univariate analysis. The combination of these two features (a
simplified definition of medullary-like type) was present in 17% of tumours and was an independent prognostic
factor on multivariate analysis. This simplified definition had good inter-observer reproducibility (=0.61) and
is worthy of more detailed assessment in an unselected group of mammary carcinomas. A fibrotic focus was
present in 36% of carcinomas. Only 3% of tumours with a fibrotic focus had features of medullary-like
carcinomas. Fibrotic focus of greater than 30% of the tumour was associated with a poor prognosis. This study
emphasizes the heterogeneity of morphology and behaviour of triple-negative basal-like carcinomas.
Breast Cancer Lung Metastasis Requires Expression of Chemokine Receptor CCR4 and
Regulatory T Cells
Purevdorj B. Olkhanud1, Dolgor Baatar1, Monica Bodogai1, Fran Hakim3, Ronald Gress3, Robin L.
Anderson2, Jie Deng1, Mai Xu1, Susanne Briest4 and Arya Biragyn1
1
Laboratory of Immunology, National Institute on Aging, Baltimore, Maryland; 2 Cancer Biology Laboratory, Peter MacCallum
Cancer Centre, Melbourne, Australia; 3 Experimental Transplantation and Immunology Branch, Bethesda, Maryland; and 4 Breast
Cancer Center, University of Leipzig, Leipzig, Germany
Requests for reprints: Arya Biragyn, National Institute on Aging, 251 Bayview Boulevard, Suite 100, Baltimore, MD 21224. Phone:
410-558-8680; Fax: 410-558-8284; E-mail: biragyna@mail.nih.gov .
Key Words: breast cancer Tregs NK cell regulation CCR4 CCL17 GBP
Cancer metastasis is a leading cause of cancer morbidity and mortality. More needs to be learned about
mechanisms that control this process. In particular, the role of chemokine receptors in metastasis remains
controversial. Here, using a highly metastatic breast cancer (4T1) model, we show that lung metastasis is a
feature of only a proportion of the tumor cells that express CCR4. Moreover, the primary tumor growing in
mammary pads activates remotely the expression of TARC/CCL17 and MDC/CCL22 in the lungs. These
chemokines acting through CCR4 attract both tumor and immune cells. However, CCR4-mediated chemotaxis
was not sufficient to produce metastasis, as tumor cells in the lung were efficiently eliminated by natural killer
(NK) cells. Lung metastasis required CCR4+ regulatory T cells (Treg), which directly killed NK cells using -
galactosidebinding protein. Thus, strategies that abrogate any part of this process should improve the outcome
through activation of effector cells and prevention of tumor cell migration. We confirm this prediction by killing
CCR4+ cells through delivery of TARC-fused toxins or depleting Tregs and preventing lung metastasis. [Cancer
Res 2009;69(14):59966004]
Original Article
Oncogene (2013) 32, 961967; doi:10.1038/onc.2012.113; published online 2 April 2012
Mammary tumor growth and pulmonary metastasis are enhanced in a hyperlipidemic mouse
model
N Alikhani1, R D Ferguson1, R Novosyadlyy1, E J Gallagher1, E J Scheinman2, S Yakar1 and D LeRoith1,2
Abstract
Dyslipidemia has been associated with an increased risk for developing cancer. However, the implicated
mechanisms are largely unknown. To explore the role of dyslipidemia in breast cancer growth and metastasis,
we used the apolipoprotein E (ApoE) knockout mice (ApoE/), which exhibit marked dyslipidemia, with
elevated circulating cholesterol and triglyceride levels in the setting of normal glucose homeostasis and insulin
sensitivity. Non-metastatic Met-1 and metastatic Mvt-1 mammary cancer cells derived from MMTV-
PyVmT/FVB-N transgenic mice and c-Myc/vegf tumor explants respectively, were injected into the mammary
fat pad of ApoE/ and wild-type (WT) females consuming a high-fat/high-cholesterol diet and tumor growth
was evaluated. ApoE/ mice exhibited increased tumor growth and displayed a greater number of spontaneous
metastases to the lungs. Furthermore, intravenous injection of Mvt-1 cells resulted in a greater number of
pulmonary metastases in the lungs of ApoE/ mice compared with WT controls. To unravel the molecular
mechanism involved in enhanced tumor growth in ApoE/ mice, we studied the response of Mvt-1 cells to
cholesterol in vitro. We found that cholesterol increased AktS473 phosphorylation in Mvt-1 cells as well as
cellular proliferation, whereas cholesterol depletion in the cell membrane abrogated AktS473 phosphorylation
induced by exogenously added cholesterol. Furthermore, in vivo administration of BKM120, a small-molecule
inhibitor of phosphatidylinositol 3-kinase (PI3K), alleviated dyslipidemia-induced tumor growth and metastasis
in Mvt-1 model with a concomitant decrease in PI3K/Akt signaling. Collectively, we suggest that the
hypercholesterolemic milieu in the ApoE/ mice is a favorable setting for mammary tumor growth and
metastasis.
Introduction
There is growing evidence that metabolic disorders such as obesity and type 2 diabetes increase breast cancer
risk.1, 2, 3, 4, 5 Moreover, it has been demonstrated that type 2 diabetes accelerates breast cancer development
independent of obesity, and this effect may be primarily mediated by hyperinsulinemia.6, 7
Both obesity and type 2 diabetes are frequently accompanied by serum lipid abnormalities such as elevated total
cholesterol, low-density lipoprotein cholesterol and triglycerides and reduced high-density lipoprotein
cholesterol, which also increase the risk of various types of human malignancies, including breast cancer. The
results of numerous epidemiological studies suggest that dysregulated cholesterol metabolism might be a key
factor linking dyslipidemia and cancer.8, 9, 10, 11 However, it remains unclear whether abnormally elevated
cholesterol levels promote tumorigenesis independent of obesity and type 2 diabetes.
Cholesterol is a sterol that serves as a metabolic precursor to other bioactive sterols and plays a major role in the
structure of the plasma membrane by creating a class of detergent-resistant microdomains called lipid rafts.12 It
has been shown that these lipid rafts possess a range of membrane-associated signaling proteins such as receptor
tyrosine kinases.13 Recent studies have demonstrated an elevated level of cholesterol in the plasma membrane of
prostate and breast tumor cells.14, 15, 16, 17 Elevations of plasma cholesterol in animal models have been shown to
cause accumulations of cholesterol in lipid rafts, leading to reduced apoptosis, activation of Akt and increased
prostate tumor growth.18 Thus, cholesterol may be contributing to tumor growth by modifying signal
transduction through effects on lipid rafts.15, 18, 19 Indeed, recent studies provide evidence that cholesterol
mediates localization of Akt molecules to lipid raft microdomains and leads to their activation.20
Epidemiological studies have described a reduced occurrence of certain malignancies in patients consuming
HMG-CoA (3-hydroxy-3-methyl-glutaryl coenzyme A) reductase inhibitors (statins) used for the treatment of
dyslipidemia.21 Statins are inhibitors of the rate-limiting step in cholesterol biosynthesis (conversion of HMG-
CoA to mevalonate). Furthermore, prostate cancer progression has recently been shown to be inhibited by a
cholesterol-lowering agent.22, 23 Although the effects of statins in breast cancer are somewhat controversial, some
studies report a lower risk of breast cancer in patients taking statins.24, 25
Here we investigated the effects of hypercholesterolemia on mammary tumor growth and metastasis, using the
apolipoprotein E (ApoE)-null mice (ApoE/). When fed a cholesterol-rich diet, ApoE/ mice show elevated
cholesterol and triglyceride levels in plasma. Interestingly, however, these mice show increased sensitivity to
insulin and have normal or low glucose levels, as has been demonstrated previously.26 We found that mammary
tumors in hypercholesterolemic ApoE/ mice were larger than those of wild-type (WT) mice and resulted in
more spontaneous pulmonary metastases.
Discussion
Here we present that hypercholesterolemia (and/or hypertriglyceridemia) without concomitant hyperglycemia or
hyperinsulinemia affects mammary tumor growth and metastasis.
The ApoE glycoprotein functions as a regulator of plasma lipid levels and participates in the uptake of lipids
into different tissues by binding to lipoprotein receptors and thereby delivering both cholesterol and
triglycerides into cells. The absence of ApoE leads to the accumulation of cholesterol and triglycerides in
plasma.27 Therefore, mice lacking the ApoE gene fed HC diet represent a well-known model for studying
atherosclerosis.27 In addition, deletion of the ApoE gene results in significantly lower levels of blood glucose
and insulin, which is most likely the consequence of an impaired lipid uptake by adipose tissue and muscle,
leading to an improvement of insulin sensitivity.26 The hypercholesterolemic phenotype found in ApoE/ mice
also makes them a useful tool for investigating the effect of elevated cholesterol levels on different types of
malignancies. The effect of upregulated triglycerides on mammary tumor growth in this model cannot be
excluded, as elevated triglyceride levels have been demonstrated in patients with different types of malignancies
including breast cancer patients.28, 29, 30 Furthermore, triglyceride-rich lipoproteins have been shown to promote
proliferation of PC-3 prostate cancer cells, potentially by influencing the regulation of lipoprotein receptor
expression.31 In addition, the role of dietary energy intake and its contribution to tumor growth in our model
cannot be excluded as obesity and high total energy intake has been reported to be associated with progression
of prostate cancer.24, 32
We used two orthotopic mammary tumor models, Met-1 (derived from MMTV-PyVmT/FVB/N transgenic
mice) and Mvt-1 (derived from c-Myc/vegf tumor explants), in ApoE/ and WT female mice. When fed regular
chow, tumor growth in ApoE/ and WT mice did not show significant differences (data not shown). However,
when fed HF/HC diet, tumors in ApoE/ mice were more aggressive, evident by increased primary tumor size
and metastatic lesions. In concordance with our data, a recent study examined the role of cholesterol in tumor
progression using the MMTV-PyMT transgenic mice. In this study, a diet-induced elevation in plasma
cholesterol levels enhanced mammary tumor development.33 Additionally, in our study, we show that the
number of pulmonary metastases is increased in the hyperlipidemic ApoE/ mouse. Similarly, growth of
prostate tumor xenografts was significantly increased in severe combined immunodeficient (SCID) mice with
elevated levels of circulating cholesterol. This growth was associated with increased cholesterol content in lipid
rafts isolated from the tumor xenografts, and was accompanied by activation of membrane-localized Akt.18
The lung is a susceptible organ to colonization by circulating tumor cells because of its large surface area and
rich blood supply.34 Upon examination of WT and ApoE/ mice harboring metastatic Mvt-1 tumors, we detected
significantly elevated numbers of spontaneous pulmonary metastases in the ApoE/ mice. However, it has been
proposed that larger tumors may distribute a greater number of metastasizing cells.34, 35 To investigate whether
increased metastases in ApoE/ mice are independent of primary tumor size, we intravenously injected an
identical number of Mvt-1 cells into ApoE/ and WT mice and quantified metastatic lesions in the lungs after 3
weeks. We found significantly more macro-metastases in ApoE/ mice, suggesting that elevated cholesterol
levels promoted the proliferation of Mvt-1 cells in the lungs. The ability of cancer cells to metastasize to a
greater degree in the hypercholesterolemic environment may be because of the formation of cholesterol-rich cell
extensions called invadopodia, which are believed to have a role in migration of cancer cells, essential for
invasion and metastasis.36
To begin investigating the molecular mechanism involved in enhanced tumor growth in the ApoE/ mice, we
studied the response of Mvt-1 cells to cholesterol in vitro. Acute cell response to cholesterol was studied using
40200mg/dl cholesterol to reproduce the physiological range of blood cholesterol in normal and
hypercholesterolemic states, respectively. Using these concentrations, we demonstrated that cholesterol
enhanced Akt activation in Mvt-1 cells. These results are in agreement with a previous study of human
epidermoid carcinoma cells treated with 40mg/dl cholesterol leading to activation of Akt.14 Furthermore,
cholesterol depletion in the cell membrane in the presence of methyl--cyclodextrin abrogated Akt
phosphorylation induced by exogenously added cholesterol. Statistical analysis was determined using analysis
of variance. Similar to our observation in vivo, we also found that cholesterol increased cellular proliferation of
Mvt-1 cells in vitro. We found that the concentration of cholesterol required to activate the Akt pathway was
much higher than that required to induce cellular proliferation. The reason for this difference is as yet
undefined, but one possibility is that to mediate the aggregation of a significant amount of lipid raft-mediated
membrane receptor signaling complexes over a short time period (2060min), much higher levels of cholesterol
are required than to induce cell proliferation over an extended time period of 72h. We also found the same
effect in MC-38 colon cancer cell line (data not shown).
The PI3K/Akt pathway plays an important role in many cancers, contributing to apoptosis, cell differentiation
and cell proliferation.37 Blocking the PI3K/Akt pathway in vivo by treating ApoE/ mice with the BKM120
compound significantly alleviated the accelerated tumor growth and metastases to the lung in comparison with
vehicle-treated mice; however, we did not observe a complete cessation of tumor growth in the BKM120-
treated mice. Therefore, these data suggest that additional signaling pathways may be involved in dyslipidemia-
induced tumor development and progression. We also see an effect of BKM120 in WT mice, likely due to
inhibition of the PI3/Akt pathway and its effect on cell proliferation.
In conclusion, we demonstrate that dyslipidemia markedly accelerates primary mammary tumor growth and
metastasis. Increased cholesterol levels are associated with enhanced phosphorylation of Akt and mammary
cancer cell growth in vitro. Furthermore, the mammary tumor-promoting effect of dyslipidemia in ApoE/ mice
is abrogated by BKM120, a specific inhibitor of the catalytic subunit of PI3K, the upstream activator of Akt. To
our knowledge, the PI3K/Akt pathway has only been shown to be cholesterol sensitive in prostate cancer. Our
investigations have indicated one potential mechanism whereby cholesterol can promote tumor growth and
metastasis in breast cancer. Our data link dyslipidemia and the PI3K/Akt pathway in mammary tumor growth
mechanistically and thus suggest that reducing total cholesterol levels may be an important therapeutic modality
in the prevention and treatment of breast cancer.
Figure 1.
Conceptual models of intratumor heterogeneity. (A) The clonal evolution model postulates that tumors
originate from a single cell; through stochastic genetic events due to increased genetic instability, new clones
emerge. The fittest clones under a set of selective pressures survive. Although a few founder genetic aberrations
are found in all cancer cells (pink star), other driver mutations (purple star and triangle and pink triangle) are
restricted to subpopulations of cancer cells within a tumor. Note that many mutations acquired throughout
evolution may either be neutral (purple four-pointed star), deleterious (pink circle) or lethal (pink four-pointed
star) to cancer cells. (B) The cancer stem cell model postulates that tumors are generated from a rare population
of cells capable of self-renewal and differentiation into different lineages. These cells give rise to the progeny of
differentiated cells, which have a limited replicative potential. Note that genetic heterogeneity has now been
documented not only in the population of differentiated cells, but also within the cancer stem cell compartment
of tumors.
In this review, the evidence of inter-and intra-tumor heterogeneity in breast cancer, recently highlighted by
massively parallel sequencing studies is discussed. Furthermore, the potential causes and implications of
intratumor genetic heterogeneity for the clinical management of breast cancer patients are reviewed and
contextualized.
Phenotypic & Genetic Intratumor Heterogeneity
Genomic and transcriptomic analyses of breast cancer have unraveled the intertumor molecular heterogeneity of
breast cancers. It is now accepted that breast cancers constitute a remarkably heterogeneous group of diseases,
[9,10]
to the extent that oestrogen receptor (ER)-positive and ER-negative breast cancers are perceived as distinct
entities that have different risk factors, clinical presentation, pathological features and clinical behavior, which
only happen to affect the same anatomical site and originate in the same microanatomical structure. [1012] The
realization of the extent of breast cancer intertumor molecular heterogeneity has led to a paradigm shift in the
way breast cancer clinical trials are designed and is changing the way breast cancer patients are managed in
clinical practice.
It should be noted, however, that the genomics studies carried out in the last decade have only constituted the
first steps in the characterization of genotypicphenotypic correlations in breast cancer, [11,12] and there is
evidence to suggest that many more are likely to emerge. The study of rare tumors that are phenotypically
homogeneous [6] has led to the identification of specific (i.e., pathognomonic) mutations. [6] For instance,
granulosa cell tumors of the ovary and adenoid cystic carcinomas affecting the breast and salivary glands are
examples of phenotypically homogenous tumors underpinned by specific genetic aberrations: mutations in
FOXL2 are found in >97% of granulosa cell tumors of the ovary [13] and the MYB-NFIB fusion gene is found in
>90% of breast adenoid cystic carcinomas and >30% of salivary gland adenoid cystic carcinomas. [14,15] At the
other end of the spectrum, common cancers harbor a wide range of mutations and even those that share several
phenotypic characteristics harbor distinct mutations. Despite extensive study of a significant number of breast
cancer cases, only a few genes were found to be highly recurrently targeted by somatic mutations (e.g., PIK3CA
and TP53), [9] exemplifying the difficulty in identifying additional driver mutations other than those in well-
known oncogenes and tumor suppressor genes in common types of malignancies. This difficulty may, in part, be
due to the lack of a standardized definition for a 'driver' mutation, the heterogeneity among breast cancers and
the lack of computational tools to accurately predict the functional relevance of observed mutations. In addition
to intertumor heterogeneity, it is becoming increasingly clear that intratumor heterogeneity will also have
important implications in early detection and treatment options. [16]
Breast cancer has long been known to display tremendous phenotypic heterogeneity, in terms of morphology,
cell surface marker expression, immunohistochemical patterns and 'stemness' properties of cancer cells, among
others. [1] For instance, assessment of HER2 expression by immunohistochemistry, not uncommonly,
demonstrates that tumors are composed of clones that differ in their phenotype. [17] Furthermore, while HER2
status between primary and metastasis is generally concordant, there are instances when this is not the case. [18,19]
An explanation for some of the phenotypic heterogeneity evident in most tumors may be the existence of
underlying genetic heterogeneity resulting from clonal evolution. [1]
The ability of breast cancers to metastasize to distant organs such as bone and the brain in untreated patients
provides the perfect setting to study heterogeneity. Given the physiological and anatomical barriers, the extent
of heterogeneity between spatially separated sites is likely to be greater than within spatially contiguous sites, in
a way akin to the phenomenon of migration and geographic isolation in Darwinian evolutionary models. Studies
involving genomic profiling of the primary breast tumors and their matched metastases have invariably
suggested that they are clonal in origin, [20] yet their genomes are not always identical. [2123] While synchronous
metastases tend to be largely the same as their corresponding primaries, [21,23] a study of 29 primaries and their
matched metachronous metastases demonstrated significant differences in gene copy number by comparative
genomic hybridization and FISH in 31% of cases. [22] The genetic diversity reported in breast cancer parallels
findings in leukemia and metastatic pancreatic and renal cancers, where subclones follow a branching clonal
evolutionary pattern. [2426] In addition, there is evidence to suggest that some distant metastases may originate
from clones present in other metastases rather than from the clones present in the primary tumor. [24] Even before
overt metastases ensue, disseminated tumor cells can often be found in the bone marrow and blood (reviewed
in.) [27] The analyses of disseminated tumor cells in bone marrow and their respective primary breast cancers
have supported the contention that dissemination may occur early. [27] Furthermore, circulating tumor cells
(CTCs) have been shown to display divergent copy number changes when compared to cells from primary
tumors, [28,29] and microsatellite analysis of CTCs from patients with multifocal prostate cancer has suggested
that CTCs are closely related to distinct, sometimes small, foci within the primary but not necessarily the
dominant clone. [30] These studies provide evidence for the hypothesis that metastatic deposits may originate
from a nonmodal clone in the primary tumor and that cells from primary cancers and their respective metastatic
deposits may undergo parallel evolution. Furthermore, these observations support the notion that in some cases,
systemic spread may be an early step in breast cancer carcinogenesis. [31] The hypothesis of early dissemination
is interesting, as this suggests that there may be substantial time for parallel evolution to occur and to generate
additional genetic diversity.
The development of genetic heterogeneity between primary tumors and their respective metastases may not,
however, always be a case of Darwinian geographical isolation. An additional layer of complexity in the
intratumor genetic heterogeneity between primary and metastasis lies in the hypothesis of 'self-seeding', which
refers to the CTCs re-entering the primary tumor. [32] By implanting a green fluorescent protein (GFP)-tagged
'donor' cell line into the mammary gland and identical but untagged 'recipient' cell line into the contralateral
mammary gland in mouse models, it was demonstrated that GFP-tagged donor tumor cells can disseminate from
the primary and 'seed' the contralateral tumor. [33] It should also be noted that more aggressive tumors appear to
be more capable of self-seeding. [33] The self-seeding phenomenon suggests that the traditional concept of a
unidirectional spread of tumor cells from primary to metastasis may not be correct. [32] This concept has
important implications for the clonal evolutionary model and may help explain the close genetic relationship
between the primary and metastatic deposits beyond what the current clonal evolutionary model offers, and the
oligoclonal nature of metastatic deposits.
Another potential barrier in the development of breast cancers is the progression from in situ to invasive
disease, [34] and genetic analyses of synchronous in situ and invasive breast cancers may provide insights into
the clonal composition and evolution of breast cancer. Invasive breast cancers are frequently associated with
multiple foci of ductal carcinoma in situ (DCIS). While DCIS is sometimes considered a precursor of their
invasive counterparts, the DCIS and the infiltrating components occasionally display distinct phenotypes and
genotypes. [35] This implies that even though the invasive component and DCIS share a common ancestor,
invasive tumors may not necessarily be derived from the modal population of neoplastic cells of the adjacent
DCIS. [36] Although previous studies were unable to find significant genetic differences between DCIS and
invasive breast cancers, [37,38] a recent study has provided evidence to suggest that some DCIS may be composed
of multiple genetically-distinct clonal populations. [35] This evolutionary pattern is perhaps better explained by
the hypothesis that the selection of nonmodal clones drives the progression from DCIS to invasive ductal
carcinoma. [35]
The recent advent of massively parallel sequencing has seen a wave of studies on the characterization of
intratumor genetic heterogeneity in numerous cancer types. [2426,3943] Two approaches (Figure 2) have been used
to quantify the extent of heterogeneity, namely single-cell sequencing and deep sequencing (). Single-cell
sequencing involves isolation of a random population of single cells from a tumor, performing whole-genome
amplification (WGA) and sequencing their amplified genomes. By flow-sorting breast tumor cells by DNA
content and sequencing the single cells at low depth to profile genome-wide copy number aberrations, it was
discovered that pseudodiploid and aneuploid subpopulations coexist in an intermingling fashion. [44]
Furthermore, the array of private copy number breakpoints identified suggests that in some breast cancers, the
pseudodiploid subpopulation is likely to be highly heterogeneous and has not undergone clonal expansion,
whereas the highly aneuploid tumor cells are likely to have resulted from one or more rounds of clonal
expansion in a 'punctuated' fashion (i.e., occasional rapid clonal expansion). [44] Single-cell exome sequencing
has recently been employed to identify nucleotide-level genetic aberrations in a myeloproliferative neoplasm
and a clear cell renal cell carcinoma (ccRCC) after isolating single cells (Figure 2). [45,46] While the
myeloproliferative neoplasm was shown to be largely monoclonal, the ccRCC consisted of a mixture of somatic
mutations at varying frequencies, suggesting that the tumor analyzed was likely to be composed of multiple
clonal nonmodal populations without a dominant clone. [45,46]
Table 1. Summary of the advantages and disadvantages of using single-cell sequencing
and deep sequencing to characterize intratumor heterogeneity.
Single-cell sequencing Deep sequencing
Library preparation is simple and standard
Very quantitative in terms of identifying the degree
of heterogeneity Putative mutations can be validated by
Advantages
orthogonal platforms
No specialist analysis methods required
Relatively cheap
Advanced statistical methods are required
Isolation of single cells is technically challengingto infer the relative frequencies of each
clonal population in a sample of mixed
DNA amplification step introduces biases caused by cells
uneven amplification of different genomic regions,
allelic dropout, amplification error and artifactual The resultant estimate of the degree of
mutations heterogeneity is semi-quantitative and is
Disadvantages
highly dependent on the statistical methods
Validation of putative mutations by orthogonal used
platforms is not possible
The identification of clones is based on
Sequencing a large number of cells can be very unsupervised approaches, whose
costly mathematical assumptions have not yet
been fully tested
Figure 2.
Approaches to the quantification of tumor heterogeneity by massively parallel sequencing. (A) Single-cell
sequencing. Isolation of single cells from a tumor can be achieved through flow sorting by DNA content or cell
dispersion with microcapillary pipetting systems. Isolated cells are then subjected to whole-genome
amplification followed by massively parallel sequencing to determine their individual mutations. (B) Deep
sequencing. A heterogeneous tumor sample is sequenced to high redundancy. Through computational methods,
deconvolution of the clonal structure is achieved and their mutational repertoire is inferred. The clonality of
different cells or cell populations can be determined by the presence (blue) or absence (gray), and prevalence of
specific mutations within the population of cancer cells sequenced.
An alternative approach to single-cell sequencing is the deep sequencing of tumors, which involves sequencing
a tumor sample containing mixtures of cells to high redundancy (Figure 2). Deep sequencing makes use of the
fact that massively parallel sequencing produces a digital, quantitative signal, which enables, through statistical
algorithms, the inference of the proportions of tumor cells within a tumor harboring a given somatic genetic
aberration and the likely clonal frequencies. [3941] In contrast to single-cell sequencing where clonal structure is
determined by the random sampling of cells prior to sequencing, inference of clonal structure is performed after
sequencing using statistical approaches. In many of the cases that have been subjected to deep sequencing,
multiple subpopulations with different genotypes were found in the cancers. [26,39,40] While some of the mutations
are shared by most of the clones, up to 52 and 69% of mutations were not found in all tumor regions in
metastatic pancreatic and renal cancers, respectively. [24,47] At the time of diagnosis, triple-negative breast
cancers were shown to harbor a wide spectrum of clonal frequencies, where only 31% of cases had three or
fewer clonal genotypes. [40]
Massively parallel sequencing studies have also revealed that the modal clonal frequency may not be stable
within a patient. In fact, not only is there direct evidence of topological clonal heterogeneity within primary
tumors, [24,44] but also there is evidence to suggest that some metastatic deposits are enriched for cancer cells
with mutations which, although present in the primary tumor, are not found in the modal population. [41] This
suggests that the process of dissemination leads to a shift in allele frequencies and, at least initially, reduction in
clonal diversity, which may reflect the selective pressures of the microenvironment and/or systemic therapies.
[41]
Seminal studies by Nik-Zainal et al. have provided fundamental clues for the understanding of the
emergence of genetic heterogeneity within a breast cancer, and the mutational mechanisms and DNA repair
defects involved in this process. [48,49] Using the principle of the most-recent common ancestor (i.e., the clone
that has the full repertoire of somatic mutations found in all cancer cells) and elegant bioinformatic algorithms,
[49]
the authors traced the entire genealogy of a breast cancer back to the fertilized egg. A crucial observation
made in those studies is that in breast cancer, contrary to previous observations made in myeloid leukemia, [50]
the most-recent common ancestor emerged rather early in tumor evolution, driver mutations precede the onset
of large scale structural and numerical chromosomal instability, and a substantial proportion of the evolution
time in a breast cancer is involved in the generation of diversity and heterogeneity. [49] Despite the genetic
heterogeneity observed in all samples, a dominant subclone comprising >50% of the cancer cells within a
sample was identified in all tumors analyzed. [49] Based on these observations, it was posited that the
development of a dominant subclone is likely to have a substantial impact on whether or not a lesion is
clinically detectable. [49]
In some cases, the genetic diversity may in part explain the phenotypic diversity often found within cancers.
Genomic analyses of microdissected morphologically distinct components of tumors have demonstrated that
morphologically distinct components of breast cancer, albeit clonal, harbor distinct genetic aberrations and that
these aberrations may underpin, or at least be coincidental with, the phenotypic diversity found in breast
cancers. [51,52] A study examining the correlation of the copy number amplification of ERBB2 (which encodes the
protein HER2) and the level of HER2 overexpression in different tissue microarray cores demonstrated
intratumor heterogeneity of HER2 amplification in up to 11% of cases. [17]
In addition to genetic heterogeneity, other mechanisms, such as intratumor epigenetic heterogeneity, are likely
to contribute to the phenomenon of intratumor phenotypic diversity. [1] A detailed discussion of intratumor
epigenetic heterogeneity is beyond the scope of this review and the readers are referred to excellent reviews on
this topic. [1,53]
Sources of Intratumor Heterogeneity
One of the prerequisites for Darwinian evolution is clonal heterogeneity (i.e., genetic heterogeneity), which
provides the substrate for selection to act on. The complete profiling of somatic mutations in tumors has enabled
the characterization of the properties, prevalence and consequences of genomic instability and its potential as a
source of heterogeneity. In fact, genomic instability is regarded as an 'enabling characteristic' that helps
acquiring mutations [54] and cells that express a mutator phenotype are more efficient at acquiring mutations than
those without a mutator phenotype. [55] At the time of diagnosis, tumors may be at various stages of 'genome
evolution' or may have very different levels of genomic instability, thus displaying a wide range of somatic
mutations. [40] As discussed above, even some DCIS (i.e., preinvasive breast cancers) display intratumor genetic
heterogeneity, [35] and tumors currently classified as of the same phenotype may differ in terms of their levels of
genetic instability and intratumor genetic heterogeneity. For instance, in the subset of triple-negative breast
cancers, basal-like breast cancers are more genetically unstable and display a greater degree of clonal
heterogeneity than nonbasal-like tumors. [40] Importantly, however, neither all basal-like breast cancers are
clonally heterogeneous nor all nonbasal-like triple-negative breast cancers are composed of a single modal
population. [55]
With the exception of cancers that clearly display a mutator phenotype (e.g., tumors harboring microsatellite
instability), the absolute number of point mutations per cell division does not deviate substantially from the
number of point mutations acquired in a single division by normal cells. [56] It should be noted, however, that
cancer cells also acquire other types of genetic aberrations, including insertions-deletions, structural
rearrangements and gene copy number aberrations. Analyses of the repertoire of somatic genetic aberrations in a
cancer provide an opportunity to define the potential mechanisms resulting in genetic instability and intratumor
genetic heterogeneity. Given that most of the somatic genetic aberrations found in a given cancer genome are
'passenger' events, detailed quantitative and qualitative analysis of the large number of passenger genetic
aberrations provides a means to define the underlying mutational processes that ultimately shape cancer
genomes. For instance, environmental exposure to carcinogens, such as ultraviolet radiation [5,57] and tobacco, [3]
leave 'footprints' in the genome, in the form of dramatically elevated mutation rates and specific nucleotide
changes.
Mutations and/or epigenetic aberrations affecting genes that maintain genome integrity also result in the
development of 'mutator' phenotypes, which can lead to markedly increased mutation rates [58] and specific
patterns of somatic mutations. In breast cancer, some of the most frequently mutated genes are in fact those
involved in checkpoint control and DNA repair. For instance, BRCA1 and BRCA2, the two genes most
frequently mutated in hereditary breast and ovarian cancer patients, play crucial roles in homologous
recombination (HR). [59] Inactivation of these genes seems to result in specific patterns of point mutations, and
microhomology-mediated indels of up to 50bp. [48,60] In addition to HR DNA repair deficiency conferred by the
loss of BRCA1 or BRCA2, a number of mutator phenotypes have been described in breast cancer, each of which
are probably underpinned by distinctive mechanisms. Stephens et al. described the phenomenon of a mutator
phenotype in breast cancers characterized by the presence of multiple intra chromosomal structural
rearrangements stemming from tandem duplications; [4] this mutator phenotype, whose molecular basis is yet to
be defined, has been shown to be more prevalent in ER-negative/HER2-negative breast cancers but not caused
by BRCA1 or BRCA2 loss of function. [4] This mutator phenotype has also been recently identified in a subset of
ovarian carcinomas, [61] and shown to not be related to BRCA1 or BRCA2 germline mutations. [61] Microsatellite
instability can be caused by defects in the mismatch repair pathway, [62] while aneuploidy, one of the most
widespread types of genomic aberration, can be caused by chromatid cohesion defects involved in chromosome
segregation. [63] Other patterns of mutator phenotypes include the accumulation of genome amplifications that
can be caused by breakage fusion bridge cycles resulting from telomere dysfunction, [2,64] and an event known as
'chromothripsis', [65] which is a catastrophic shattering of a chromosome or an arm of a chromosome and the
subsequent random religation of the fragments, resulting in tens to hundreds of genomic rearrangements
occurring in a one-off cellular crisis. This phenomenon has been documented in approximately 25% of
osteosarcomas and in a small subset of breast cancers, [65] including tumors arising in BRCA1 germline mutation
carriers. [60]
More recently, five new mutational signatures characterized by specific patterns of base pair changes have been
identified in breast cancer samples: [48] mutational signature A, characterized by C>T transitions at CpG sites;
mutational signature B, which is more complex and involves C>T transitions preferentially at TpCpA and
TpCpT sites, C>G mutations predominantly at TpCpA and TpCpT sites, and C>A mutations at TpCpA and
TpCpT sites; mutational signature C, which is characterized by C>T and C>G mutations at CpG sites;
mutational signature D, which comprised a uniform distribution of the different mutational classes; and
mutational signature E, whose dominant feature was C>G mutations at TpCpX trinucleotides, but lacks the C>T
mutations at TpCpX trinucleotide characteristics of mutational signature B. [48] Furthermore, a new type of
catastrophic mutational event, named kataegis (from the Greek 'shower' or 'thunderstorm') was discovered. This
catastrophic event is characterized by incredibly high numbers of somatic base substitutions, preferentially
cytosine at TpC dinucleotides, clustered in regions comprising hundreds of bases (i.e., microclusters) or
millions of bases (i.e., macroclusters). [48] Interestingly, kataegis may take place together with chromothripsis.
[48,65]
The mechanisms underpinning these signatures have yet to be fully characterized, however it is plausible
that APOBEC1 and APOBEC3 enzymes, which play roles in the deamination of cytosine residues in B
lymphocytes triggering hypermutation and an innate antiretroviral defense, may be involved in the genesis of
the mutational signature B and/or kataegis. [48]
Genetic instability, however, is not necessarily caused by genetic alterations in genes that control genomic
integrity. In fact, epigenetic events such as hypermethylation, chromatin remodeling and histone modifications,
may result in increased genetic instability. [66] Hypermethylation of p16 INK4a ( CDKN2A) has been found in
histologically normal mammary epithelia, [67] suggesting a role in early carcinogenesis. Interestingly, epigenetic
events sometimes result in inactivation of pathways that are crucial for the maintenance of genomic integrity.
For example, promoter methylation of MLH1 leading to microsatellite instability in a subset of nonfamilial (i.e.,
sporadic) colorectal cancer. [68] Similar to genetic instability, epigenetic instability can be caused by the loss of
function in genes that maintain the integrity of the epigenome. [66,69] The CpG island methylator phenotype is
well described for colorectal cancer, [70] but remains to be fully elucidated in other solid malignancies. [71]
Upregulation of DNA methyltransferase-3B ( DNMT3B) has been proposed as one of the mechanisms of CpG
island methylator phenotype in both colorectal and breast cancers. [72,73] However, recent studies have also
implicated a genetic basis for epigenetic aberrations in cancer. Rearrangements and mutations have been
identified in a number of genes involved in chromatin remodeling, such as ARID1A and PBRM1, in ovarian
clear-cell carcinoma and renal carcinoma, [7,8] respectively, suggesting that the SWI/SNF chromatin remodeling
complex is an important component in maintaining epigenetic stability. As for breast cancer, mutations in
EP300, a histone acetyltransferase, and BRG1, a component of the SWI/SNF complex, have been documented.
[74,75]
It is likely that epigenetic instability contributes to the breast cancer intratumor phenotypic heterogeneity.
Another potential explanation for the phenotypic heterogeneity found in cancers is the cancer stem cell (CSC)
hypothesis (Figure 1B), which postulates that there exists a rare population of cells that display stem cell-like
behavior, including the ability to self-renew, to divide asymmetrically and to differentiate into different lineages,
[76]
giving rise to the phenotypic diversity found in cancers. CSCs are classically defined by their phenotypic and
functional properties. By isolating putative CSCs based on the expression of a specific set of surface markers,
followed by limited diluting assays and transplantation into immunocompromised mice, putative CSC
populations were first identified in leukemias. [77] Since then, similar methods have been used to identify
putative CSCs in solid malignancies, including breast cancer, where a combination of cell surface markers, such
as CD44 +/CD24 /low cells [78,79] and ALDH1-positive cells, [80] has been employed for the identification of CSCs.
The existence, characteristics and biological significance of CSCs have proven controversial in solid tumors.
First, although CSCs are identified by cell surface markers, the 'gold-standard' assay to define 'stemness' is
based on xenotransplantation of subpopulations of human cancer cells into nude mice. [76] By changing the level
of immunosuppression in the recipient mice, up to 25% of cells within melanomas could be considered to have
stem-like properties, questioning the fundamental premise of the 'rare' stem cell theory. [81] Second, the
discovery of the dedifferentiation of mammary epithelial cells into stem-like cells casts doubt about the
differentiation hierarchy that the CSC model proposes. [82] Third, in some cases, putative breast CSCs and
nonstem cells appeared to be genetically divergent suggesting that the differentiated cells may not have
necessarily derived directly from the so-called stem cells. [79,83]
The CSC and the clonal evolution models are, however, not necessarily mutually exclusive as putative CSC
populations are not necessarily genetically homogeneous. By serial transplantation into mice, there is direct
evidence to suggest that considerable genetic diversity exists within a leukemic propagating cell population.
[25,42]
Comparison of the degree of subclonal genetic heterogeneity in the original patient samples to that in the
xenografts transplanted into multiple mice, demonstrates that the genetic diversity within the propagating cell
populations mirrors that of the primary tumor at the subclonal level. [25] Similar findings have been made in
breast cancer where combined immunofluorescence staining and FISH experiments suggest chromosomal
aberrations were variable within the stem cell-like CD44 + breast cancer cell population. [83] These studies in
leukemia and breast cancer suggest that the original CSC model may be overly simplistic [84] and that multiple
genetically distinct subpopulations within the so-called CSC compartment are likely to exist. The level of
stemness of genetically different clones of CSCs has yet to be fully characterized. Interestingly, recent data from
complete genome sequencing of 21 breast genomes suggested that the most recent cancer ancestor clones within
a tumor appear to constitute a relatively long-lived population of quiescent cells; these characteristics would be
reminiscent of those of the so-called CSCs. [49]
Clinical Implications of Intratumor Heterogeneity
The existence of intratumor genetic and phenotypic heterogeneity has important implications in clinical
management. Genetic heterogeneity suggests that there may be subclones within a tumor that manage to evade
treatment, and if this occurs, these subclones may grow out to cause relapse (otherwise referred to as 'acquired
resistance'). [16] However, in most circumstances, the term 'acquired resistance' is perhaps misleading, as tumor
cells with intrinsic resistance mutations are often pre-existing in a minor subclone of the tumor, and are selected
for when systemic therapies are applied. [16,8587] As the resistance phenotype is not selectively advantageous in
the absence of chemotherapy, one determinant of the probability of the presence of a pre-existing resistant
subclone may be the extent of heterogeneity. The degree of clonal diversity has been found to be a good
estimate of the likelihood of response to chemoradiotherapy in cervical cancer [88] and can predict the likelihood
of progression from a premalignant lesion to esophageal carcinoma. [89]
Some of the strongest evidence for the contribution of genetic heterogeneity to acquired resistance comes from
studying resistance mechanisms to targeted therapies. Imatinib mesylate, a small molecule inhibitor that targets
ABL, is the mainstay of treatment of chronic myeloid leukemia (CML) harboring the pathognomonic fusion
gene BCR-ABL. Although response rates to imatinib are high at 5 years, approx 6% of patients progress to the
accelerated phase and 3% have a hematological relapse. [90] Resistance has been shown to be caused by either
mutations in the ABL kinase domain, such as p.T315I, p.Q252H/R and p.Y253F/H, [85] which either alter amino
acids that directly contact imatinib or prevent BCR-ABL from achieving the inactive conformational state
required for imatinib binding, or amplification of the BCR-ABL fusion gene. [91] In fact, these secondary
mutations were often not, secondary but were present in minor subclones at diagnosis. [85,92] Although these
mutations have been shown to result in resistance to imatinib, there is evidence to demonstrate that mutations in
the kinase domain sometimes do not grow out to be the dominant clone in relapse, [93] suggesting that multiple
mechanisms of resistance may cooperate. Furthermore, in some patients, resistance to imatinib is polyclonal,
with multiple secondary BCR-ABL mutations being detected in a subset of patients with CML relapse post-
imatinib treatment. [85]
Similar observations have been made in gastrointestinal stromal tumors (GIST) and non-small cell lung cancer
(NSCLC). While all cells within GIST harbor the same founding mutation in KIT, imatinib selects for the clones
with secondary KIT mutations, [94] which prevent the binding of the drug to the catalytic domain and cause the
outgrowth of tumor. In a way akin to CML, imatinib-resistant lesions may contain more than one secondary
KIT mutation. [95] Likewise, NSCLC may develop resistance to anti-EGF receptor (EGFR) by the outgrowth of
clones harboring secondary EGFR mutations that prevent the binding of anti-EGFR inhibitors to the EGFR
catalytic domain, such as T790M, or the amplification of MET. [86,87]
On a genome-wide scale, several studies have shown that relapses descend from a minor subclone surviving
initial chemotherapy. [25,41,43] Copy number analysis of matched diagnosis and relapse samples from pediatric
acute lymphoblastic leukemia cases identified divergent evolutionary patterns. [43] Intriguingly, backtracking
analysis of relapse-specific genomic breakpoints in diagnosis samples suggests that 52% of relapses originate
from an ancestral subclone that predates diagnosis. [43] Two separate sequencing studies of breast cancer
comparing the brain metastases to the original primary by deep massively parallel sequencing revealed that the
clones that constituted the modal populations in the metastases were present in the primary lesions, but in the
form of minor nonmodal clones, with prevalence as low as 1%. These observations suggest clonal selection
driven by chemotherapy and, potentially, by the steps required in metastatic dissemination and adaptation to a
distinct microenvironment. [39,41]
As evidence points to the extent and effect of genomic and epigenomic instability in cancers, from an
evolutionary standpoint, targeting the cause of the genetic/epigenetic instability may constitute an approach to
circumvent genetic heterogeneity within cancers. Selectively targeting tumor cells with HR and mismatch repair
pathways defects by the alternative DNA repair pathway and creating synthetic lethality has been shown to be a
promising approach. [6,96,97] Clinical studies have shown that poly(ADP) ribose polymerase (PARP) inhibitors
lead to sustained responses in breast and ovarian cancer patients harboring germline BRCA1 or BRCA2
mutations. [9698] For colorectal cancers with microsatellite instability caused by an MSH2 defect, in vitro studies
have shown that methotrexate may be effective in targeting cells with MSH2 loss of function. [99] Some have
proposed that tumor taxonomies ought to be based on their genetic defects and patterns of genetic instability
rather than the site of origin, [100] as supported by the good response of HR deficient patients to PARP inhibitors,
regardless of tissue origin. [9698] However, even in the case of targeting the driving genetic events causative of
the genetic instability found in cancers, resistance has been shown to develop. For instance, resistance to
platinum salts and PARP inhibitors has been shown to be caused by the selection of subclones of cancer cells
harboring either an intragenic deletion that removes the initial mutant, or a secondary mutation that restores the
BRCA1 or BRCA2 reading frame in cancers from patients harboring BRCA1 or BRCA2 germline mutations,
respectively. [101104] The occurrence of secondary mutations in BRCA1 and BRCA2 in breast and ovarian
cancers, in KIT in GIST and in EGFR in NSCLC highlights the importance of cataloging potential resistance
mechanisms and to develop drugs that would target escape mechanisms. [16]
As many epithelial cancers harbor thousands of mutations and the most common cancers probably involve
many different genetic routes to malignancy, [6] intratumor heterogeneity may provide a means to pinpoint driver
mutations and to reconstruct evolutionary history. [24,44,49] Mutations that are present ubiquitously within a tumor
are more likely to have been in the founding clone than mutations that are present only in a subpopulation. [40]
Single-cell exome sequencing of a ccRCC reveals 66 mutations with low mutation frequencies ('hills'), with
only 28 mutations with high mutation frequencies ('mountains'), of which AHNAK was of particular interest as a
candidate driver gene as it was found to be mutated with high frequencies in the index case and to be recurrently
mutated in approx 5% of 99 ccRCCs. [46] Another recent study, by sampling and sequencing multiple sites from
renal carcinomas, reconstructed the evolutionary history and confirmed that mutations in VHL in renal
carcinomas occur early in carcinogenesis. [24] The study also revealed that two distinct regions of the same tumor
harbored different mutations in each of SETD2, a histone methyltransferase, and KDM5C, a histone H3K4
demethylase. [24] Convergent evolution suggests that mutations in these two genes, both involved in histone
modification, are either the result of strong selective pressures or essential for the survival of this tumor at that
stage of evolution. This type of approach may lead to the identification of the mutated genes that may constitute
optimal targets for tumor debulking, and the identification of subclones that drive resistance to specific
therapeutic interventions.
The current stage of technological development allows for unraveling the clonal structure of cancers, which
may provide the basis for improvement in the design of individualized treatment. It is, in theory, plausible to
identify combinations of therapeutic agents that would target every detectable subclone such that tumor burden,
and hence tumor cell population size, is reduced to a minimum to reduce the chances of the emergence of
resistant clones. [105] This would mark a significant step forward in cancer medicine. The rapid development and
decreasing costs of massively parallel and single-cell sequencing will provide a maturing platform for the
potentials of personalized medicine to be realized. For an in-depth discussion of the clinical implications of
intratumor genetic heterogeneity in the management of cancer, readers are referred to Turner and Reis-Filho. [16]
Expert Commentary
Intratumor genetic heterogeneity presents significant challenges to the successful clinical management of
cancers. The advancements in sequencing technology have enabled the comprehensive study of cancer
genomes, both in terms of the complete catalog of mutations in a given tumor at a subclonal level and the
characterization of genomic instability as a driving force in cancer evolution. A better understanding of
intratumor genetic heterogeneity and the processes by which it is generated will enable more effective
approaches for the implementation of individualized therapy.
The extent of intratumor heterogeneity observed in various cancer types, including breast cancers and ccRCCs,
suggests that a paradigm shift in the way systemic therapies are delivered is required: rather than considering
that each patient with cancer harbors one tumor, it would perhaps be worth considering that each patient harbors
multiple genetically distinct malignancies with private genetic aberrations that may render them resistant to
specific systemic therapies that target the cancer model population. While personalized medicine involving the
use of targeted therapy has revolutionized cancer medicine, its full potential is unlikely to be realized until the
concept of intratumor genetic heterogeneity is factored in. [16,106]
The effectiveness of rationally designed tailored treatment hinges on our ability to profile intratumor genetic
and phenotypic heterogeneity comprehensively. It is plausible that the extent of heterogeneity has been vastly
underestimated. Key challenges to overcome this hurdle include, first, changing the current routine practice of
single biopsy from patients, which does not allow the genotyping of distant metastases or even tumor cell
populations in other areas within a primary tumor. Tissue collection at multiple tissue sites where possible,
regular blood collection for the harvesting of CTCs and tissue collection at relapse would allow more
comprehensive profiling of heterogeneity and a more complete understanding of potential resistance
mechanisms. Second, while the recent studies of single-cell genome and exome sequencing [4446] have paved the
way for its wider application, this approach faces various technical challenges. Its reliance on WGA is likely to
result in biases in the estimation of heterogeneity, which cannot necessarily be corrected by validating the
results using orthogonal platforms. A thorough characterization of the biases WGA introduces will be required
before single-cell sequencing is used in the clinical arena. Third, the deconvolution of clonal structure from
deep sequencing data is an open research question and requires sophisticated statistical and computational
algorithms to be developed, where several untested assumptions are made. This is not a trivial research question
to answer and will require, in particular, a definition of what constitutes a 'clone' and what constitutes a 'relevant
clone' in evolutionary terms.
In the last decade, the concept of intertumor molecular heterogeneity has been brought to the forefront of cancer
research, and constitutes one of the lynchpins of personalized medicine. The concept of intratumor genetic
heterogeneity and its impact on the development of rationally defined combinatorial therapies, however, are yet
to be fully understood. Furthermore, understanding the basis of genetic and phenotypic heterogeneity in cancers
may provide new avenues for the development of targeted therapies that circumvent the challenges posed by the
existence of multiple clonal populations within a cancer.
Five-year View
With the plummeting costs of massively parallel sequencing, the development of more robust bioinformatic
approaches to define the repertoire of mutations, gene copy number aberrations, structural variations and
transcriptomic changes in cancers, and the reporting of the results of the large sequencing exercises carried out
by The Cancer Genome Atlas and the International Cancer Genome Consortium, [107] it is anticipated that the
complete repertoire of somatic genetic aberrations found in the modal populations of cancer cells from common
cancer types will be characterized in the next 5 years. It should be noted, however, that this will only be the
starting point. Given the levels of intratumor genetic heterogeneity and the current design of the Cancer
Genome Atlas and International Cancer Genome Consortium projects, it is probable that mutations of biological
importance, but present in submodal populations of cancer cells may not be identified; hence, additional and
optimally designed sequencing endeavors, to address in greater detail the relevance of mutations found in
submodal populations of cancer cells, will be of paramount importance. Studies to distinguish between driver
and passenger genetic aberrations will also be required, which are by no means trivial and will require a
combination of bioinformatic approaches and extensive functional genomics experiments. Furthermore, the
unraveling of epistatic interactions involving driver and passenger mutations will be crucial for the development
of rational combinatorial therapies, [6] as will be the understanding of the interactions between germline genetic
variants and somatic mutations. [108] Finally, the characterization of the predatory and mutualistic interactions
between distinct subclones of cancer cells and between cancer cells and their microenvironment [1,16,53] may
prove essential for the successful implementation of personalized medicine.
Sidebar
Key Issues
Tumors evolve by acquiring 'driver' and 'passenger' mutations, which contribute to intratumor genetic
heterogeneity.
Intratumor phenotypic heterogeneity can be, in part, underpinned by genetic differences.
Massively parallel studies have shown that clonal heterogeneity is common in breast cancers and the
level of heterogeneity varies between cases that are currently classified into the same clinical subtypes.
Acquired resistance to targeted therapy is commonly driven by the selection of nonmodal populations of
cancer cells harboring secondary mutations in the target gene.
Targeting the causes of genetic and epigenetic instability may be a means to circumvent heterogeneity.
To realize the potentials of personalized medicine, drug combinations ought to be rationally designed
based on the repertoire of genetic aberrations found in the modal and nonmodal populations of a given cancer.
Single-cell sequencing presents a means to explore the biological and clinical implications of intratumor
genetic heterogeneity.
Biological Differences in Male vs Female Breast Cancer
September 15, 2011 Men are on average older than women when diagnosed with breast cancer and have
differences in disease characteristics compared with their female counterparts, confirmed a new study presented
at the 2011 Breast Cancer Symposium in San Francisco, California.
"Men are diagnosed with breast cancer at an older age and more frequently have lymph node involvement at
diagnosis compared to women," said lead author Siva K. Talluri, MD, in an interview with Medscape Medical
News. He is a clinical assistant professor in the McLaren Internal Medicine Residency Program at Michigan
State University in Flint, Michigan.
The fact that men present later in life and with more advanced disease than women "may be related possibly to
the lack of awareness among patients as well as primary care physicians and absence of screening routinely
done in women," Dr. Talluri said.
Dr. Talluri and colleagues conducted a retrospective cohort study that included information on 2475 men and
393,259 women with breast cancer from the National Cancer Institute's Surveillance, Epidemiology, and End
Results (SEER) database.
Median age at diagnosis was 67 years for men vs 61 for men. Lymph node involvement was present in 32% of
men and 22% of women, and men were more likely to be estrogen receptor (ER) positive or progesterone
receptor (PR) positive than women.
In men with breast cancer, overall median survival duration was 9 years, the 5-year survival rate was 63%, and
the 10-year survival rate was 43%. Factors associated with decreased survival were age older than 65 years at
diagnosis, larger tumor size, positive lymph node status, ER-negative status, and poorly differentiated grade (P
= 0.02). PR status was not a significant predictor of survival.
Median survival was significantly shorter in African-American men with breast cancer than in white men (7.08
vs. 9.2 years; P = .02).
"We updated the information on survival and predictors of male breast cancer by analyzing more recent data
from 1990-2007," said Dr. Talluri. "There is a paucity of epidemiologic data on male breast cancer because it so
rare; therefore any information garnered from such large datasets as SEER is valuable." She added that presence
of ER-negative status was not an independent predictor in previous studies but was significant in this study.
Dr. Talluri added that the study was missing some potentially important data. "We did not have information
about our study population on the risk factors like BRCA, and family history of breast cancer. I am interested to
know about risk factors that will help us identify the men at high risk. This may help the clinicians to diagnose
men with breast cancer at an earlier stage of the disease."
Gail S. Lebovic, MD, a member of the 2011 Breast Cancer Symposium News Planning Team and past president
of the American Society of Breast Disease, commented, "While the study itself has some limitations, the authors
confirm what has been shown historically. Breast cancer in men occurs later in life, is frequently associated with
a delay in diagnosis, and is commonly associated with lymph node involvement. Although breast cancer is rare
in men, these findings demonstrate that it is critically important to continue to raise awareness about the
occurrence of breast cancer in men."
Dr. Talluri and Dr. Lebovic have disclosed no relevant financial relationships.
2011 Breast Cancer Symposium; abstract #39. Presented September 8, 2011.