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British Journal of Cancer (2012) 107, 864873. doi:10.1038/bjc.2012.347 www.bjcancer.

com
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.

British Journal of Cancer (2012) 107, 874878. doi:10.1038/bjc.2012.337 www.bjcancer.com


Published online 26 July 2012
Coffee and black tea consumption and breast cancer mortality in a cohort of Swedish women
H R Harris1,2, L Bergkvist3 and A Wolk1
Background:
Coffee and black tea contain a mixture of compounds that have the potential to influence breast cancer risk and
survival. However, epidemiologic data on the relation between coffee and black tea consumption and breast
cancer survival are sparse.
Methods:
We investigated the association between coffee and black tea consumption and survival among 3243 women
with invasive breast cancer in the Swedish Mammography Cohort. Intake was estimated using a food frequency
questionnaire. Cox proportional hazard models were used to calculate hazard ratios (HRs) and 95% confidence
intervals (95% CIs).
Results:
From 1987 to 2010 there were 394 breast cancer-specific deaths and 973 total deaths. Coffee and black tea were
not associated with breast cancer-specific or overall mortality. Women consuming 4+ cups of coffee per day
had a covariate and clinical characteristics-adjusted HR (95% CI) of death from breast cancer of 1.14 (0.71
1.83; ptrend=0.81) compared with those consuming <1 cup per day. Women consuming 2+ cups of black tea per
day had a covariate and clinical characteristics-adjusted HR (95% CI) of death from breast cancer of 1.02
(0.671.55; ptrend=0.94) compared with non-tea drinkers. Caffeine was also not associated with breast cancer-
specific (HR for top to bottom quartile=1.06; 95% CI=0.791.44; ptrend=0.71) or overall mortality.
Conclusion:
Our findings suggest that coffee, black tea, and caffeine consumption before breast cancer diagnosis do not
influence breast cancer-specific and overall survival.

Breast Cancer Cells Can Turn off Key Immune


Response: Study
By Tan Ee Lyn
HONG KONG (Reuters) Jul 23 - Breast cancer cells can shut down a powerful immune response, which allows
the disease to spread to the patient's bones, researchers in Australia reported on Monday.
They also experimented with two ways to reinstate this immune response to help patients fight breast cancer,
but it will take more tests and several more years for these therapies to become routine treatments, they said.
"We have identified a way that breast cancer cells can turn off the immune system, allowing them to spread to
distant parts such as the bone," said the study's senior author Belinda Parker, a research fellow at the Peter
MacCallum Cancer Centre in Melbourne.
"By understanding how this occurs, we hope to use existing and new therapies to restore this immune function
and prevent the spread of cancer," she said by telephone.
The study was published Monday in the journal Nature Medicine.
Using tissue samples from breast cancer patients and experiments with mice, Parker and colleagues found that a
gene called IRF7 is switched off in patients whose cancer spreads to other parts of the body.
IRF7 controls the production of interferon, an important immune signaling protein that fights viruses and
bacteria apart from tumor cells
"Usually when breast cancer cells leave the breast and travel in the bloodstream and into bone marrow, the
release of interferons by IRF7 will cause the immune system to recognize those cells and eliminate them,"
Parker said.
"But by losing IRF7, it prevents the stimulation of immune responses and allows those cells to hide from being
recognized (and later spread)."
Parker and her team tried two ways to revive this immune response in experiments with mice and both appeared
to work.
"We put the gene back into cancer cells so it can't switch it (IRF7) off. We allowed the immune pathway to be
stimulated and the cancer cells did not spread to the bone," Parker said.
"The other way is to treat the animals with interferon, which is available for treating other diseases, like
hepatitis. That also prevented the spread of cancer to the bone."
Parker said they will study how best to use these two methods on patients in the next few years and plan to have
a clinical trial in two to three years.
SOURCE: http://bit.ly/Oj3XZ2 Nature Medicine, online July 22, 2010.

NOTCH-1 and NOTCH-4 are Novel Gene Targets of


PEA3 in Breast Cancer
Novel Therapeutic Implications
Anthony G Clementz; Allison Rogowski; Kinnari Pandya; Lucio Miele; Clodia Osipo
Posted: 09/01/2011; Breast Cancer Research. 2011;13(3):R63 2011 BioMed Central, Ltd.
Abstract and Introduction
Abstract
Introduction: Women with triple-negative breast cancer have the worst prognosis, frequently present with
metastatic tumors and have few targeted therapy options. Notch-1 and Notch-4 are potent breast oncogenes that
are overexpressed in triple-negative and other subtypes of breast cancer. PEA3, an ETS transcription factor, is
also overexpressed in triple-negative and other breast cancer subtypes. We investigated whether PEA3 could be
the critical transcriptional activator of Notch receptors in MDA-MB-231 and other breast cancer cells.
Methods: Real-time PCR and Western blot analysis were performed to detect Notch-1, Notch-2, Notch-3 and
Notch-4 receptor expression in breast cancer cells when PEA3 was knocked down by siRNA. Chromatin
immunoprecipitation was performed to identify promoter regions for Notch genes that recruited PEA3. TAM-67
and c-Jun siRNA were used to identify that c-Jun was necessary for PEA3 enrichment on the Notch-4 promoter.
A Notch-4 luciferase reporter was used to confirm that endogenous PEA3 or AP-1 activated the Notch-4
promoter region. Cell cycle analysis, trypan blue exclusion, annexin V flow cytometry, colony formation assay
and an in vivo xenograft study were performed to determine the biological significance of targeting PEA3 via
siRNA, Notch signaling via a -secretase inhibitor, or both.
Results: Herein we provide new evidence for transcriptional regulation of Notch by PEA3 in breast cancer.
PEA3 activates Notch-1 transcription in MCF-7, MDA-MB-231 and SKBr3 breast cancer cells. PEA3 activates
Notch-4 transcription in MDA-MB-231 cells where PEA3 levels are endogenously high. In SKBr3 and BT474
breast cancer cells where PEA3 levels are low, overexpression of PEA3 increases Notch-4 transcripts.
Chromatin immunoprecipitation confirmed the enrichment of PEA3 on Notch-1 and Notch-4 promoters in
MDA-MB-231 cells. PEA3 recruitment to Notch-1 was AP-1-independent, whereas PEA3 recruitment to
Notch-4 was c-JUN-dependent. Importantly, the combined inhibition of Notch signaling via a -secretase
inhibitor (MRK-003 GSI) and knockdown of PEA3 arrested growth in the G1 phase, decreased both anchorage-
dependent and anchorage-independent growth and significantly increased apoptotic cells in vitro. Moreover,
either PEA3 knockdown or MRK-003 GSI treatment significantly reduced tumor growth of MDA-MB-231
xenografts in vivo.
Conclusions: Taken together, the results from this study demonstrate for the first time that Notch-1 and Notch-4
are novel transcriptional targets of PEA3 in breast cancer cells. Targeting of PEA3 and/or Notch pathways
might provide a new therapeutic strategy for triple-negative and possibly other breast cancer subtypes.
Introduction
Breast cancer continues to be the second leading cause of cancer-related deaths among women worldwide.
Approximately 70% of breast cancers are estrogen receptor -positive (ER+) and progesterone receptor-
positive (PR+). They are divided into two subtypes: luminal A, comprising those that are negative for the
overexpression or gene amplification of ErbB-2/HER2 and have low levels of genes responsible for
proliferation, and luminal B, comprising those that are positive for HER2 and have high expression of
proliferation-associated genes.[1,2] This division is in part due to their sensitivity to antihormonal therapy such as
tamoxifen or an aromatase inhibitor. The luminal A subtype carries the best prognosis, followed by luminal B.
The third subtype is ER/PR and HER2+, which contains gene amplification for the ErbB-2/HER2 oncogene.
The HER2+ subtype represents 15% to 25% of breast cancers and is currently treated with trastuzumab plus a
taxane-based chemotherapy. The HER2+ subtype of breast cancer is associated with excellent survival outcomes
due to adjuvant trastuzumab therapy, a humanized monoclonal antibody that targets the HER2 receptor.[3]
However, 30% to 60% of metastatic HER2+ breast cancer are resistant to trastuzumab. The fourth subtype of
breast cancer is the normal-like subtype, which resembles normal mammary epithelial cells expressing genes
associated with adipose tissue. The fifth subtype represents 15% of breast cancers and is triple-negative, and
thus lacks expression of ER/PR and HER2. Triple-negative breast cancers carry the worst prognosis because of
the lack of US Food and Drug Administration-approved targeted therapies.[4,5] Thus, there is an immediate need
for the elucidation of novel targets to treat women with triple-negative breast cancer and to increase these
patients' overall survival.
Notch signaling has emerged as a target for the treatment of breast cancer.[6] In the mammalian system, there are
four Notch receptors (Notch-1, Notch-2, Notch-3 and Notch-4)[7] and five known ligands (Delta-like 1, Delta-
like 3 and Delta-like 4 and Jagged-1 and Jagged-2).[810] Cell-to-cell contact is critical for the activation of Notch
signaling, which subsequently enables the pathway to modulate genes involved in cell fate such as proliferation
or differentiation.[11] Notch is processed in the trans-Golgi apparatus, where it undergoes the first of three
proteolytic cleavages. The single polypeptide is cleaved (S1) by furin-like convertase forming the mature Notch
receptor, which is a heterodimer consisting of Notch extracellular (NEC) and Notch transmembrane (NTM).
The receptor is trafficked to the plasma membrane, where it awaits engagement with its membrane-associated
ligand. Upon ligand-receptor engagement, the second cleavage (S2) by a disintegrin and metalloproteases 10
and 17 (ADAM10 and ADAM17, respectively) [12] releases NEC to be endocytosed into the ligand-expressing
cell. Subsequently, NTM is cleaved (S3) by the -secretase complex, liberating the intracellular portion of
Notch (NIC).[13] NIC translocates to the nucleus and binds to CBF-1, a constitutive transcriptional repressor,
displacing corepressors and recruiting coactivators such as Mastermind.[14,15] Notch activates many genes
associated with differentiation and/or survival, including, but not limited to, the HES and HEY family of basic
helix-loop-helix transcription factors,[16] cyclin D1[17] and c-Myc.[18] The third and final cleavage step is critical
for active Notch signaling. Its inhibition can be exploited through emerging pharmacological drugs identified as
-secretase inhibitors (GSIs), which attenuate signaling from all four receptors. Recent studies have
demonstrated that GSI treatment suppresses breast tumor growth in a variety of breast cancer subtypes, [1923]
providing evidence of novel therapeutic approaches.
The first evidence that Notch receptors are breast oncogenes was provided by mouse studies. Overexpression of
constitutive, active forms of Notch-1 (N1IC) or Notch-4 (N4IC) form spontaneous murine mammary tumors in
vivo.[11] Furthermore, elevated expression of Notch-1 and/or its ligand Jagged-1 in human breast tumors is
associated with the poorest overall patient survival.[2426] Recently, Notch-4 has been shown to be critical for the
survival of tumor-initiating cells.[27] Similarly to studies performed using Notch-1, mouse mammary tumor virus
(MMTV)-driven Notch-3 receptor intracellular domain expression in transgenic mice showed enhanced
mammary tumorigenesis.[28] In HER2 breast cancers, downregulation of Notch-3 resulted in suppressed
proliferation and increased apoptosis.[29] In contrast, overexpression of Notch-2 in MDA-MB-231 cells
significantly decreased tumor growth and increased apoptosis in vivo,[30] suggesting that Notch-2 is a breast
tumor suppressor.
The factors that regulate Notch receptor expression in breast cancer cells are still widely unknown. It has been
shown that p53 binds to the Notch-1 promoter and activates Notch-1 receptor transcription in human
keratinocytes.[31] Activator protein 1 (AP-1) has been demonstrated to be a transcriptional activator of Notch-4
in human vascular endothelial cells.[32] We asked which factors regulate Notch receptor transcription in breast
cancer.
Polyomavirus enhancer activator 3 (PEA3/E1AF/ETV4) is a member of the ETS family of transcription factors,
which also includes ERM and ER-81. PEA3 is overexpressed in metastatic breast carcinomas, particularly
triple-negative breast tumors.[33] PEA3 regulates critical genes involved in inflammation and invasion, such as
IL-8,[34] cyclooxygenase-2 (COX-2)[35] and matrix metalloproteases (MMPs).[3639] A dominant-negative form of
PEA3 reduced tumor onset and growth in a MMTV/neu-transgenic model of breast cancer in vivo.[40] PEA3
contains an ETS winged helix-turn-helix DNA binding motif[41] that binds to the canonical sequence GGAA/T
on target genes.[42] The affinity of binding relies on proximal sequences surrounding the ETS binding site which
aid in transcriptional control based on context.[43] Phosphorylation of serine and threonine residues by the
mitogen-activating protein kinase cascade activates PEA3 and is negatively regulated by the ubiquitin-
proteasome pathway as well as by sumoylation.[4446]
The transcriptional activity of PEA3 is dependent on other activators to regulate gene transcription and is
commonly partnered with AP-1 to regulate genes such as MMP-1, MMP-3, MMP-7 and MMP-9;[36] urokinase-
type plasminogen activator (uPA);[47] COX-2;[35] and ErbB-2.[48] AP-1 is a dimeric complex consisting of the Fos
(c-FOS, FosB, Fra-1 and Fra-2) and Jun (c-JUN, JunB and JunD) families.[49] Depending on the cellular context,
AP-1 cooperates with other proteins including, but not limited to, NFB, CBP/p300, Rb and PEA3.[50,51] The
functional role of AP-1 is to recruit and direct appropriate factors to regulate gene expression and promote
proliferation, differentiation, inflammation and/or apoptosis.[52]
Previous investigations have determined that overexpression of Notch-1 and Notch-4 plays a critical role in
breast tumorigenesis[11] and that PEA3 overexpression is associated with aggressive breast cancers, particularly
the triple-negative subtype.[5358] Herein we provide novel evidence of a link between two pathways that are
overexpressed in breast cancer. PEA3 is a transcriptional activator of Notch-1 and Notch-4 and a repressor of
Notch-2 in MDA-MB-231 cells, an example of triple-negative breast cancer cells. PEA3-mediated Notch-1
transcription is AP-1-independent, while Notch-4 transcription requires both PEA3 and c-JUN. PEA3 and/or
Notch signaling are essential for proliferation, survival and tumor growth of MDA-MB-231 cells. Furthermore,
PEA3 is a transcriptional activator of both Notch-1 and Notch-4 in other breast cancer cells. Thus we
hypothesized that targeting of the PEA3 and/or Notch pathways might provide a new therapeutic strategy for
triple-negative breast cancer as well as possibly other breast cancer subtypes where PEA3 regulates Notch-1
and/or Notch-4.
Discussion
PEA3 was originally identified as a member of the ETS family of transcription factors. Since then, it has been
observed that PEA3 is expressed during normal breast development, is quickly lost upon maturation and yet
reemerges in metastatic breast cancers.[33,54,61,6668] Similarly, Notch has been implicated in breast cancer,
demonstrating elevated expression and activity in breast tumors.[25,26] Little is known about the factors that
influence Notch gene expression and why its levels are elevated in breast cancer. Herein we provide the first
evidence of transcriptional regulation of Notch-1 and Notch-4 by PEA3 in MDA-MB-231 and other breast
cancer subtypes (Figures 1 through 3). We have demonstrated that Notch-4 gene regulation is dependent on AP-
1 factors such as c-Jun, Fra-1, c-Fos and now PEA3, depending on cellular context (Figures 5 and 7).
Interestingly, we provide evidence that c-Jun and Fra-1 are transcriptional activators of Notch-4, but that c-FOS
could be a transcriptional repressor of Notch-4 (Figure 7). PEA3 regulates the Notch-1 promoter, but the
additional factors that aid in that regulation are still being investigated. Further evidence reveals that PEA3
inhibition helps sensitize MDA-MB-231 cells to a GSI, showing promise in significantly reducing both
anchorage-dependent and anchorage-independent growth as well as increasing apoptosis in vitro (Figures 8, 9A
and 9B). Moreover, PEA3 expression or Notch activity is required for optimal growth of MDA-MB-231 tumors
in vivo (Figure 9C). Thus, PEA3 emerges as a potential target, possibly upstream of Notch activity, for triple-
negative cancer and possibly other breast cancer subtypes where PEA3 and/or Notch activities are critical for
growth. This was evident in the preclinical model of our MDA-MB-231 xenograft study (Figure 9C). Either
PEA3 knockdown or GSI treatment was adequate to significantly inhibit tumor growth in vivo. This result could
suggest that targeting PEA3, a transcriptional activator of Notch-1 and Notch-4, hits multiple targets at once.
For example, PEA3 is an activator of MMPs,[3639,53] uPAR,[55,56] COX-2 [35] and now Notch-1 and Notch-4. PEA3
expression and Notch signaling could be critical for tumor formation and communication with the tumor
microenvironment, which is not possible to recapitulate in vitro using single-cell suspensions. The severe side
effects associated with GSI treatment, such as gastrointestinal toxicity,[69,70] could possibly be avoided if
inhibition of PEA3 is able to inhibit several growth- and metastasis-promoting signaling pathways.
Notch is a cellular fate determinant and can induce cell proliferation and/or differentiation, depending on the
cellular environment.[71] PEA3 has been linked to the invasion, migration and aggressiveness of tumor cells.
[33,40,53,54,57,61,67,68,72,73]
The dual or individual inhibition of Notch by the GSI, and the inhibition of PEA3 by siRNA,
acts by preventing two vital arms of cancer progression, namely, growth and possibly invasion, which we are
currently investigating in vitro and in vivo. Emerging nanotechnology can be used as a means by which to direct
siRNA therapies,[74] and the advent of stapled interface peptides[75] that disrupt transcription factor complexes
transforms the notion of specific targeting of the PEA3 transcription factor into a potential reality.
In addition, Harrison et al.[27] implicated Notch-4 in mammary tumor stem cell survival and self-renewal in a
recent study in which they demonstrated that targeting Notch-4 specifically was more effective than a targeting
a GSI in inhibiting the Notch pathway. In our studies, we found that Notch-4 gene transcription was more
sensitive to PEA3 inhibition. Given this fact, the sensitivity that we obtained in our MDA-MD-231 system by
the dual inhibition using a PEA3 siRNA in combination with MRK-003 GSI reduced viability and increased
apoptosis may be explained by the notion that we may have targeted not only the proliferation and survival of
bulk cancer cell populations but also possibly the cancer stem cell population.
Herein we have provided evidence of a novel therapeutic strategy to be exploited for the treatment of triple-
negative breast cancer and potentially other breast cancer subtypes where PEA3 regulates Notch-1 and Notch-4.
Enhanced sensitivity toward current GSIs or alternative strategies for future clinical trials by inhibition of PEA3
by nanoparticles, small-molecule inhibitors or future siRNA approaches may increase patient response to
treatment and could reduce or eliminate recurrence if stem cell populations are eliminated. Inhibiting PEA3 may
also allow for a larger therapeutic window for GSI treatment, enabling the reduction of pharmacological doses
or possibly eliminating the need for the GSI if PEA3 is indeed upstream of Notch signaling, thus lowering
resultant undesirable side effects such as gastrointestinal toxicity and possibly skin cancer.
Conclusions
Taken together, the results from this study demonstrate for the first time that Notch-1 and Notch-4 are novel
transcriptional targets of PEA3 in breast cancer cells. PEA3 emerges as a potential innovative target upstream
from Notch activity for triple-negative cancer and possibly other breast cancer subtypes where PEA3 and/or
Notch activities are critical for growth and aggressive phenotypes. The significance of targeting of PEA3 and/or
Notch pathways allows a potentially novel therapeutic strategy for the treatment of breast cancers.

Psychosocial Stress Linked to Aggressive Breast Cancer


September 23, 2011 (Washington, DC) Stress is associated with the aggressiveness of breast cancer,
according to a study presented here at the Fourth American Association for Cancer Research Conference on the
Science of Cancer Health Disparities.
The cross-sectional study looked at the potential relation between stress and tumor aggressiveness in the human
population. Researchers found that patients with greater levels of fear, anxiety, or social isolation were more
likely to have aggressive tumors.
"What we found, very generally, was that there was an association," Garth Rauscher, PhD, associate professor
of epidemiology at the School of Public Health at University of Illinois at Chicago, told Medscape Medical
News. "Patients who reported greater levels of stress were more likely to have high-grade, more aggressive
tumors. They were also more likely to have tumors that lack hormone receptors."
During an interview 2 to 3 months after diagnosis, black and Hispanic patients with breast cancer reported
higher levels of stress than white patients, Dr. Rauscher said.
The study looked at 989 patients recently diagnosed breast cancer: 397 non-Hispanic whites, 411 non-Hispanic
blacks, and 181 Hispanics.
The researchers used 3 different stress scales, combined into a single measure of psychosocial stress. They
defined breast cancer aggressiveness in terms of hormone (estrogen and progesterone) receptor-negative tumors
and tumors with high histologic grades.
Earlier studies looked at stress and the incidence of breast cancer on survival and recurrence, but this is the first
population-based study to look at the potential role of stress in tumor aggressiveness, Dr. Rauscher said. He
noted that studies have shown that the incidence of breast cancer rises among socially isolated rat mothers.
Anil Sood, MD, professor and director of the Blanton-Davis Ovarian Cancer Research Program in the
Departments of Gynecologic Oncology and Cancer Biology at the University of Texas M.D. Anderson Cancer
Center in Houston, told Medscape Medical News that he found the study interesting because it seems to support
the concept that stress can influence cancer biology. However, he also noted that causality is difficult to
determine, given the constraints of the study design.
"For causality, one would have to see a strong relationship that can be manipulated by an intervention that
would reverse the harmful factor," he said. Because Dr. Rauscher's study is correlative, it is difficult to establish
causality or directionality, Dr. Sood explained.
Dr. Rauscher acknowledged the study's limitations. "This is not in any way a definitive study," he said, noting
that it is still a work in progress. "It's purely exploratory."
A major limitation of the study, he said, is that the stress levels are reported after diagnosis and during
treatment. As a result, it is not clear what is driving the association between stress and tumor aggressiveness. It
is completely conceivable that somebody with a more aggressive tumor might subsequently report higher levels
of stress. A woman with a more aggressive tumor who is aware of the more worrisome diagnosis would likely
feel more stress. The more aggressive treatment someone with an aggressive tumor faces might also influence
the stress she feels.
It is possible the direction of the causal relation is the opposite of what one might assume, Dr. Rauscher said.
Although the researchers were interested in the way stress influences tumor aggressiveness, it is possible they
were measuring the way tumor aggression influences stress. "There is no way in this study to disentangle them,"
Dr. Rauscher said.
A major assumption of the analysis was that patients who reported greater psychosocial stress at an interview,
which was 2 to 3 months after diagnosis, would have also reported relatively greater psychosocial stress had
they been interviewed before diagnosis.
Dr. Rauscher noted that although the association between stress and tumor aggression shrank a bit after
adjustment for variables such as stage of diagnosis and status of treatment at the time of the interview, such
variables don't seem to account for the relations they saw.
According to Dr. Rauscher, the potential role of DNA methylation, as it relates to race and ethnicity and tumor
aggression, is worth exploring.
He also said it would make sense to go back and revisit some of the earlier cohort studies that used a variety of
stress measures, and look to see if they have medical records and pathology data on these tumors. It might help
to see if researchers can identify which ones were more adverse and less aggressive and tease out the role of
stress. "It's very hard to study," he said. "How do you even measure stress?"
Drs. Rauscher and Dr. Sood have disclosed no relevant financial relationships.
Fourth American Association for Cancer Research (AACR) Conference on the Science of Cancer Health
Disparities (SCHD): Abstract A91. Presented September 19, 2011.

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.

Bone Drug Improves Breast Cancer Survival


June 7, 2011 Zoledronic acid, combined with anastrozole or tamoxifen, led to improved disease-free survival
rates in women with stage I or stage II breast cancer, according to a study published online June 4 in The Lancet
Oncology. The results suggest that zoledronic acid could be an important addition to adjuvant endocrine therapy
in premenopausal patients with early-stage breast cancer.
The report outlined an analysis of the Austrian Breast and Colorectal Cancer Study Group trial 12, which is a
randomized controlled, open-label multicenter trial. It included 1803 premenopausal women diagnosed with
endocrine-receptor-positive breast cancer (stage I or stage II). All patients received goserelin (3.6 mg every 28
days).
Separate groups compared the efficacy and safety of anastrozole (1 mg/day) or tamoxifen (20 mg/day), with or
without zoledronic acid (4 mg every 6 months). Participants were followed up for 3 years. Patients were
randomly assigned to the 4 groups in a 1:1:1:1 ratio across prognostic variables that included age, neoadjuvant
chemotherapy, pathological tumor stage, lymph node involvement, type of surgery or locoregional therapy,
complete axillary dissection, intraoperative radiation therapy, and geographical region.
Median follow-up time was 62 months (range, 0 - 114.4 months). At least 2 years after treatment completion,
there were a total of 186 disease-free survival events reported (53 events in 450 patients receiving tamoxifen
alone, 57 in 453 patients receiving anastrozole alone, 36 in 450 patients receiving tamoxifen plus zoledronic
acid, and 40 in 450 patients receiving anastrozole plus zoledronic acid).
Zoledronic acid was associated with a reduction in the risk for disease-free survival events (hazard ratio [HR],
0.68; 95% confidence interval [CI], 0.51 - 0.91; P = .009).The differences were not statistically significant for
the tamixofen (HR, 0.67; 95% CI, 0.44 - 1.03; P = .067) and anastrozole (HR, 0.68; 95% CI, 0.45 - 1.02; P = .
061) groups when they were analyzed individually.
Zoledronic acid had no influence on death (30 deaths with zoledronic acid vs 43 deaths without; HR, 0.67; 95%
CI, 0.41 - 1.07; P = .09). Disease-free survival was the same in patients taking tamoxifen alone compared with
patients taking anastrozole alone (HR, 1.08; 95% CI, 0.81 - 1.44; P = .591). Overall survival was lower in
patients receiving anastrozole compared with patients receiving tamoxifen (46 vs 27 deaths; HR, 1.75; 95% CI,
1.08 - 2.83; P = .02).
The treatment regimens had no associated reports of renal failure or osteonecrosis of the jaw. Of the
participants, 601 patients reported bone pain (33%; 349 patients receiving zoledronic acid vs 252 patients not
receiving zoledronic acid), 361 reported fatigue (20%; 192 vs 169), 280 reported headaches (16%; 147 vs 133),
and 266 reported arthralgia (15%; 145 vs 121).
"Addition of zoledronic acid improved disease-free survival in the patients taking anastrozole or tamoxifen,"
conclude lead author, Michael Gnant, MD, from the Medical University of Vienna, Austria, and colleagues.
"There was no difference in disease-free survival between patients receiving anastrozole and tamoxifen overall,
but those on anastrozole alone had inferior overall survival. These data show persistent benefits with zoledronic
acid and support its addition to adjuvant endocrine therapy in premenopausal patients with early-stage breast
cancer."
The study provides some insight into which patients could most benefit from zoledronic acid, according to
Joanne Mortimer, MD, director of the women's cancer program at City of Hope, Duarte, California.
"[Zoledronic acid] clearly has some benefit in some populations of women. The next step is figuring out who
that population is. This study suggests that women who have low estrogen levels are the ones that benefit most
from the bone treatment," Dr. Mortimer told Medscape Medical News.
The study received support from Novartis (zoledronic acid) and AstraZeneca (anastrozole, tamoxifen goserelin).
The study authors have consulted for Novartis, AstraZeneca, Genomic Health, and Pfizer, as well as received
lecture fees, travel awards, research support, and other compensation from Novartis, AstraZeneca, Sanofi-
Aventis, Roche, Amgen, Pfizer, GlaxoSmithKline, and Schering. Dr. Mortimer has served as a consultant to
Novartis in the past.
Lancet Oncol. Published online June 4, 2011. Abstract

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.
Top of page
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.
Top of page
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.
Top of page
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.
Top of page
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.
Top of page
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.
Top of page
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.

Surgery. 1993 Oct;114(4):637-41; discussion 641-2.

Metastatic patterns of invasive lobular versus


invasive ductal carcinoma of the breast.
Borst MJ, Ingold JA.
Source
Department of Surgery, William Beaumont Hospital, Royal Oak, Mich. 48073-6769.
Abstract
BACKGROUND:
Many studies have analyzed the metastatic patterns of breast carcinoma. However, very few studies have
analyzed the differences in metastatic patterns of lobular versus ductal carcinoma.
METHODS:
By use of our tumor registry, the metastatic sites of all invasive lobular and invasive ductal breast carcinoma
cases during an 18-year period (January 1973 to December 1990) were analyzed.
RESULTS:
There were 2605 cases of invasive lobular and invasive ductal breast carcinoma. Lobular carcinoma accounted
for 359 (14%) and ductal carcinoma for 2246 (86%) of the cases. The percentage of patients with regional
lymph node metastasis at diagnosis was not significantly different between the two groups. The rates of
metastasis to all lymph nodes, liver, and central nervous system were not significantly different. However, the
rates of metastasis to the gastrointestinal system (4.5% vs 0.2%), gynecologic organs (4.5% vs 0.8%),
peritoneum-retroperitoneum (3.1% vs 0.6%), adrenal glands (0.6% vs 0%), bone-marrow (21.2% vs 14.4%),
and lung-pleura (2.5% vs 10.2%) were significantly different (p < 0.05).
CONCLUSIONS:
The metastatic patterns of lobular and ductal carcinoma of the breast are different, with gastrointestinal system,
gynecologic organ, and peritoneum-retroperitoneum metastases markedly more prevalent in lobular carcinoma.
Physicians should be aware of these different metastatic patterns of lobular and ductal carcinoma of the breast.

Metastatic Lobular Carcinoma of the Breast


Patterns of Spread in the Chest, Abdomen, and Pelvis on CT
1. Corinne B. Winston1,
2. Orna Hadar2,
3. Jerrold B. Teitcher1,
4. James F. Caravelli3,
5. Nancy T. Sklarin4,
6. David M. Panicek3 and
7. Laura Liberman3
+ Author Affiliations
1
1. Department of Radiology, Memorial Sloan Kettering Cancer Center, Weill Medical College, Office
#862, 160 E. 53rd St., New York, NY 10022.
2
2. Pro Health Radiology, 2800 Marcus Ave., Lake Success, NY 11042.
3
3. Department of Radiology, Memorial Sloan Kettering Cancer Center, 1275 York Ave., New York, NY
10021.
4
4. Breast Oncology Service, Memorial Sloan Kettering Cancer Center, 205 E. 64th St., New York, NY
10021.

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.

Analysis: Cover Story - Cancer


Lou, K.-J. SciBX 4(25); doi:10.1038/scibx.2011.699
Published online June 23 2011
Origins of metastasis in breast cancer
by Kai-Jye Lou, Staff Writer
Researchers at the Albert Einstein College of Medicine of Yeshiva University and the Centocor Ortho Biotech
Inc. unit of Johnson & Johnson have linked multiple prometastatic processes in breast cancer to the production
of the chemokine CCL2 in tumor cells.1 The findings could aid the development of new therapeutics to prevent
breast cancer metastasis, the main cause of breast cancer mortality in Western women, 2 and could point to a new
indication for the company's CNTO 888.
CNTO 888, an antibody against human monocyte chemoattractant protein-1 (MCP-1; CCL2), is in Phase II
testing to treat metastatic, castration-resistant prostate cancer and Phase I trials for solid tumors. The compound
also is in Phase II testing to treat idiopathic pulmonary fibrosis.3
CCL2 signals through CC chemokine receptor 2 (CCR2; CD192) and has been identified as a therapeutic target
in breast cancer.4, 5 Signaling through this chemokine-receptor pair has been associated with multiple processes
that promote tumor progression and metastasis, like macrophage recruitment into the tumor microenvironment.
However, the origin of these metastasis-associated macrophages in breast cancer was unclear. Moreover, the
mechanistic details on how CCL2 promotes macrophage recruitment into the tumor microenvironment and how
the macrophages themselves promote metastasis in breast cancer have not been well defined.
Now, researchers from Albert Einstein College of Medicine and J&J have used a suite of mouse models and
cellular assays to show that CCL2, secreted from breast tumor cells disseminating from the primary malignancy,
recruits CCR2+ inflammatory monocytes. These monocytes in turn recruit and differentiate into metastasis-
associated macrophages. Finally, the macrophages promote tumor cell extravasation and metastatic seeding in a
VEGF-Adependent manner.
In multiple mouse models of human breast cancer, both CNTO 888 and a mAb targeting Ccl2 significantly
decreased the frequency of pulmonary metastasis and increased survival compared with saline (p<0.001 for
both). The anti-CCL2 mAbs also blocked tumor cell migration in vitro compared with a control antibody
(p<0.001 for anti-Ccl2 mAb; p<0.01 for CNTO 888).
Conditional ablation of Vegf-a in inflammatory monocytes or bone marrowderived macrophages inhibited the
metastatic seeding of mouse mammary cancer cells in the lung. That finding suggests that combining a CCL2
inhibitor with a VEGF-A inhibitor could have an even stronger antimetastatic effect than either compound
alone.
Results were published in Nature.
Top of page
Preventing migration
Jeffrey Pollard, corresponding author on the paper and deputy director of the Albert Einstein Cancer Center at
the university, said the study suggests that inhibitors of monocyte recruitment and function could have a
significant impact on prometastatic processes in breast cancer.
Such a therapy can be realized, for example, through anti-CCL2 antibodies and antimacrophage treatments,
he told SciBX. Our work also demonstrates a dynamic interaction between tumor cells and nontumor cells that
promotes metastasis.
What this study suggests is that rather than deplete the macrophages directly, what one might want to do is
prevent these cells from migrating to the tumor and sites of metastasis, added Amy Fulton, a professor in the
Department of Pathology at the University of Maryland School of Medicine and the University of Maryland
Greenebaum Cancer Center. The theory is interesting and sound based on the known biology of these cells.
CCL2 is one of the normal signaling molecules used by macrophages and other cells to migrate to various parts
of the body.
In addition, Pollard noted that although CCL2 had a mosaic-like expression pattern in the primary tumor, the
chemokine was expressed throughout metastatic lesions. This finding suggests that CCL2-expressing tumor
cells may be the subpopulation that preferentially extravasate from the primary tumor and migrate to metastatic
sites, he said.
If correct, this could help identify patients at increased risk for developing metastatic disease who would be
candidates for an anti-CCL2 therapy.
From our experimental data, we believe that extravasation is one of the key rate-limiting steps for the
development of metastatic lesions in breast cancer, Pollard told SciBX.
The consensus among researchers contacted by SciBX is that a CCL2-targeting therapy would most likely be
evaluated in the clinic as part of combination therapy.
There are already many different treatments for breast cancer, so it will be important to determine how a
CCL2-targeting compound could be integrated with such treatments and whether there are synergistic or
antagonistic interactions between them, said Leisha Emens, an associate professor of oncology at The Sidney
Kimmel Comprehensive Cancer Center at The Johns Hopkins University. Most treatments for breast cancer
work by targeting the tumor cells themselves, but the CCL2-targeting approach would uniquely affect an
interaction that these cells have with the tumor microenvironment.
Most treatments for breast cancer work by targeting the tumor cells themselves, but the CCL2-targeting
approach would uniquely affect an interaction that these cells have with the tumor microenvironment.
Leisha Emens
The Sidney Kimmel Comprehensive Cancer Center at The Johns Hopkins University
What one might want to consider is combining an anti-CCL2 or antimacrophage therapy with chemotherapy to
see if it results in an enhancement of therapeutic outcomes such as improving survival, said Pollard.
Kristiina Vuori, president of the Sanford-Burnham Medical Research Institute and director of its cancer center,
added that another logical duo would be a CCL2-targeting therapy and an anti-VEGF drug.
Most of the experiments performed in the current study were designed to test the hypothesis that blocking
CCL2 can inhibit early processes in breast cancer metastasis, but this may not be representative of its use in the
clinical setting, where cancer cell dissemination may have already occurred, Vuori added. They will need to
determine the timing of the effect of the CCL2-targeting antibody. It will be important to determine whether
such a therapy would be effective after metastasis has been established in distant sites.
Fulton noted that chemokine receptors can be activated by multiple ligands and that breast cancer cells
themselves express multiple chemokine receptors linked to prometastatic processes.
Because of this high degree of redundancy in the chemokine system, I think it's good to be skeptical on
whether blocking any single target in the chemokine system will have a long-term therapeutic effect, said
Fulton. While such treatments may show an initial therapeutic effect, the cancer may quickly develop an
escape mechanism. It will be important to carry out longer-term studies to determine if the breast cancer will
start using another chemokine receptor or ligand to spread.
Indeed, a 2003 study has shown that MCP-2 (CCL8) and MCP-3 (CCL7) also are ligands for CCR2.6
Pollard said his group is focused on elucidating additional details of the metastatic cascade in breast cancer.
Although the current study looked primarily at breast cancer that metastasized to the lung, Pollard said the
group is studying whether other common sites of metastasis in breast cancer use the CCL2-mediated pathway.
According to Pollard, his group has not filed for a patent on the findings described in the paper.
In 2008, J&J filed a patent application covering anti-CCL2 antibodies and their uses in multiple indications
including cancer. The company did not disclose whether it plans to develop CNTO 888 for metastatic breast
cancer.
Top of page
References
1. Qian, B.-Z. et al. Nature; published online June 8, 2011; doi:10.1038/nature10138 | Article |
Contact: Jeffrey W. Pollard, Albert Einstein College of Medicine of Yeshiva University, New York, N.Y.
e-mail: pollard@aecom.yu.edu
2. Weigelt, B. et al. Nat. Rev. Cancer 5, 591602 (2005) | Article | PubMed | ISI | ChemPort |
3. Schaeffer, S. BioCentury 19(26), A13A15; June 20, 2011
4. Soria, G. & Ben-Baruch, A. Cancer Lett. 267, 271285
(2008) | Article | PubMed | ISI | ADS | ChemPort |
5. Lu, X. & Kang, Y. J. Biol. Chem. 284, 2908729096 (2009) | Article | PubMed | ChemPort |
6. Vande Broek, I. et al. Br. J. Cancer 88, 855862 (2003)

British Journal of Cancer (2011) 104, 14011409. doi:10.1038/bjc.2011.88 www.bjcancer.com


Published online 22 March 2011
Effective treatment of chemoresistant breast cancer in vitro and in vivo by a factor VII-
targeted photodynamic therapy
Background:
The purpose of this study was to test a novel, dual tumour vascular endothelial cell (VEC)- and tumour cell-
targeting factor VII-targeted Sn(IV) chlorin e6 photodynamic therapy (fVII-tPDT) by targeting a receptor tissue
factor (TF) as an alternative treatment for chemoresistant breast cancer using a multidrug resistant (MDR)
breast cancer line MCF-7/MDR.
Methods:
The TF expression by the MCF-7/MDR breast cancer cells and tumour VECs in MCF-7/MDR tumours from
mice was determined separately by flow cytometry and immunohistochemistry using anti-human or anti-murine
TF antibodies. The efficacy of fVII-tPDT was tested in vitro and in vivo and was compared with non-targeted
PDT for treatment of chemoresistant breast cancer. The in vitro efficacy was determined by a non-clonogenic
assay using crystal violet staining for monolayers, and apoptosis and necrosis were assayed to elucidate the
underlying mechanisms. The in vivo efficacy of fVII-tPDT was determined in a nude mouse model of
subcutaneous MCF-7/MDR tumour xenograft by measuring tumour volume.
Results:
To our knowledge, this is the first presentation showing that TF was expressed on tumour VECs in
chemoresistant breast tumours from mice. The in vitro efficacy of fVII-tPDT was 12-fold stronger than that of
ntPDT for MCF-7/MDR cancer cells, and the mechanism of action involved induction of apoptosis and
necrosis. Moreover, fVII-tPDT was effective and safe for the treatment of chemoresistant breast tumours in the
nude mouse model.
Conclusions:
We conclude that fVII-tPDT is effective and safe for the treatment of chemoresistant breast cancer, presumably
by simultaneously targeting both the tumour neovasculature and chemoresistant cancer cells. Thus, this dual-
targeting fVII-tPDT could also have therapeutic potential for the treatment of other chemoresistant cancers.

British Journal of Cancer (2011) 104, 14721477. doi:10.1038/bjc.2011.122 www.bjcancer.com


Published online 5 April 2011
Circulating tumour cells in the central and the peripheral venous compartment in patients
with metastatic breast cancer
D J E Peeters1, G G Van den Eynden1, P-J van Dam1, A Prov1, I H Benoy2, P A van Dam1, P B Vermeulen1,
P Pauwels3, M Peeters4, S J Van Laere1 and L Y Dirix1
Background:
The enumeration of circulating tumour cells (CTC) has prognostic significance in patients with metastatic breast
cancer (MBC) and monitoring of CTC levels over time has considerable potential to guide treatment decisions.
However, little is known on CTC kinetics in the human bloodstream.
Methods:
In this study, we compared the number of CTC in both 7.5ml central venous blood (CVB) and 7.5ml peripheral
venous blood (PVB) from 30 patients with MBC starting with a new line of chemotherapy.
Results:
The number of CTC was found to be significantly higher in CVB (median: 43.5; range: 04036) than in PVB
(median: 33; range: 04013) (P=0.001). When analysing samples pairwise, CTC counts were found to be
significantly higher in CVB than in PVB in 12 out of 26 patients with detectable CTC. In contrast, only 2 out of
26 patients had higher CTC counts in PVB as compared with CVB, whereas in 12 remaining patients no
significant difference was seen. The pattern of CTC distribution was independent of the sites of metastatic
involvement.
Conclusion:
A substantial difference in the number of CTC was observed between CVB and PVB of patients with MBC.
Registration of the site of blood collection is warranted in studies evaluating the role of CTC assessment in
these patients.

British Journal of Cancer (2011) 104, 14781481. doi:10.1038/bjc.2011.115 www.bjcancer.com


Breast, ovarian, and endometrial malignancies in systemic lupus
erythematosus: a meta-analysis
S Bernatsky1,2, R Ramsey-Goldman3, W D Foulkes4, C Gordon5 and A E Clarke2,6
Background:
An increased lymphoma risk is well documented in systemic lupus (SLE). Less attention has been focused on
women's cancers, even though SLE affects mostly females. Our objective was to estimate the risk of breast,
ovarian, and endometrial cancers in SLE, relative to the general population.
Methods:
Data were included from five recent studies of large SLE cohorts. The number of cancers observed was
determined for each cancer type. The expected number of malignancies was ascertained from general
population data. The parameter of interest was the standardised incidence ratio (SIR), the ratio of observed to
expected malignancies.
Results:
The five studies included 47325 SLE patients (42171 females) observed for 282553 patient years. There were
376 breast cancers, 66 endometrial cancers, and 44 ovarian cancers. The total number of cancers observed was
less than that expected, with SIRs of 0.76 (95% CI: 0.69, 0.85) for breast cancer, 0.71 (95% CI: 0.55, 0.91) for
endometrial cancer, and 0.66 (95% CI: 0.49, 0.90) for ovarian cancer.
Conclusions:
Data strongly support a decreased risk of breast, ovarian, and endometrial cancers in SLE. This may be due to
inherent differences in women in SLE (vs the general population) regarding endogenous oestrogen, other
medications, and/or genetic make-up.

Interplay Between Neural-cadherin and Vascular


Endothelial-cadherin in Breast Cancer
Progression
Maryam Rezaei, Katrin Friedrich, Ben Wielockx, Aleksandar Kuzmanov, Antje Kettelhake, Myriam Labelle,
Hans Schnittler, Gustavo Baretton, Georg Breier
Breast Cancer Res. 2012;14(6):R154
Abstract and Introduction
Abstract
Introduction: Deregulation of cadherin expression, in particular the loss of epithelial (E)-cadherin and gain of
neural (N)-cadherin, has been implicated in carcinoma progression. We previously showed that endothelial cell-
specific vascular endothelial (VE)-cadherin can be expressed aberrantly on tumor cells both in human breast
cancer and in experimental mouse mammary carcinoma. Functional analyses revealed that VE-cadherin
promotes tumor cell proliferation and invasion by stimulating transforming growth factor (TGF)- signaling.
Here, we investigate the functional interplay between N-cadherin and VE-cadherin in breast cancer.
Methods: The expression of N-cadherin and VE-cadherin was evaluated by immunohistochemistry in a tissue
microarray with 84 invasive human breast carcinomas. VE-cadherin and N-cadherin expression in mouse
mammary carcinoma cells was manipulated by RNA interference or overexpression, and cells were then
analyzed by immunofluorescence, reverse transcriptase-polymerase chain reaction, and western blot.
Experimental tumors were generated by transplantation of the modified mouse mammary carcinoma cells into
immunocompetent mice. Tumor growth was monitored, and tumor tissue was subjected to histological analysis.
Results: VE-cadherin and N-cadherin were largely co-expressed in invasive human breast cancers. Silencing of
N-cadherin in mouse mammary carcinoma cells led to decreased VE-cadherin expression and induced changes
indicative of mesenchymal-epithelial transition, as indicated by re-induction of E-cadherin, localization of -
catenin at the cell membrane, decreased expression of vimentin and SIP1, and gain of epithelial morphology.
Suppression of N-cadherin expression also inhibited tumor growth in vivo, even when VE-cadherin expression
was forced.
Conclusions: Our results highlight the critical role of N-cadherin in breast cancer progression and show that N-
cadherin is involved in maintaining the malignant tumor cell phenotype. The presence of N-cadherin prevents
the re-expression of E-cadherin and localization of -catenin at the plasma membrane of mesenchymal
mammary carcinoma cells. N-cadherin is also required to maintain the expression of VE-cadherin in malignant
tumor cells but not vice versa. Thus, N-cadherin acts in concert with VE-cadherin to promote tumor growth.
Introduction
Cadherins are a family of transmembrane proteins that, together with their associated intracellular catenins,
have important functions in cell-cell adhesion. Different cell types express different members of the cadherin
family. Epithelial (E)-cadherin is a key component of adherens junctions in epithelial cells and functions as a
suppressor of tumor growth and invasion. Perturbation of its function leads to an invasive phenotype in many
tumors.[13] Neural (N)-cadherin is expressed in neural tissues and fibroblasts, where it mediates a less stable and
more dynamic form of cell-cell adhesion.[14] Vascular endothelial (VE)-cadherin is the primary component of
endothelial cell adherens junctions and has an important function in regulating vascular permeability and
angiogenesis.[5] Because of the important role played by cadherins in cell recognition, adhesion, and signaling,
modulation of their function and expression has significant implications for the progression of tumors.[1,610] For
instance, a switch from E-cadherin to N-cadherin expression contributes to increased tumor cell migration,
invasion and metastasis.[810] Aberrant expression of VE-cadherin was first detected in aggressive melanoma
cells and in some cases of sarcoma.[1113] A recent study from our group has revealed that VE-cadherin is
expressed aberrantly in a subset of tumor cells in human breast cancer.[7] In a mouse mammary carcinoma
model, VE-cadherin expression was induced in cancer cells that had undergone epithelial-mesenchymal
transition (EMT). Functional experiments showed that VE-cadherin promotes malignant tumor cell proliferation
and invasion by enhancing the protumorigenic transforming growth factor-beta (TGF-) pathway. However, the
functional interaction between VE-cadherin and N-cadherin during tumor progression is poorly characterized to
date.
EMT was first described by Elizabeth Hay in the 1980s as a central process in early embryonic morphogenesis.
[14]
The initial step of EMT includes the loss of epithelial markers such as E-cadherin via its transcriptional
repression and the gain of mesenchymal markers such as vimentin. As a consequence, the cadherin-binding
partner -catenin can dissociate from the E-cadherin complex at the plasma membrane and translocate to the
nucleus where it participates in EMT signaling and activates genes involved in tumor progression.[15] Epithelial
cells then lose their typical baso-apical polarization as cell-cell junctions disassemble. Additionally, the
cytoskeleton undergoes dynamic cortical actin remodeling and gains the front-rear polarization that facilitates
cell movement.[16] Finally, cell-matrix adhesion changes as proteolytic enzymes such as matrix metalloproteases
are activated.[17,18] The transition from an epithelial to mesenchymal phenotype is reversible; for example,
several rounds of EMT and mesenchymal-epithelial transition (MET) occur during development as cells
differentiate and the complex three dimensional structure of internal organs forms.[19] There is increasing
evidence that EMT also facilitates the dissemination of tumor cells to form distant metastasis.[20] Various
publications have described a switch between the epithelial and mesenchymal phenotypes through EMT and
MET in models of colorectal,[21] bladder,[22] ovarian[23] and breast cancer.[24] These findings indicate that the
phenotypic conversion of tumor cells in the metastatic cascade is multifaceted, with EMT being critical for the
initial transformation from benign to invasive carcinoma and the spreading of tumor cells, but MET occurring at
the site of metastatic colonization.[6]
The mouse mammary carcinoma model that we have previously used to study the expression of cadherins[7]
utilizes tumor cell lines that represent different stages of tumor progression: Ep5 cells are tumorigenic
mammary epithelial cells transformed by the v-Ha-Ras oncogene, whereas Ep5ExTu cells, isolated from Ep5
cell tumors grown in mice, have undergone EMT in vivo and present a mesenchymal, invasive and angiogenic
phenotype.[25,26] We observed that VE-cadherin expression is induced in these murine breast cancer cells during
(TGF--mediated) EMT.[7] On the other hand, E-cadherin expression was downregulated, and N-cadherin levels
remained unchanged. Silencing VE-cadherin expression inhibited tumor cell proliferation and invasion in vitro,
and experimental tumor growth in mice. However, the role of N-cadherin and its potential interaction with VE-
cadherin in this model is unclear. Here, we investigate the influence of N-cadherin on EMT and tumor
progression in Ep5ExTu cells. Silencing N-cadherin significantly decreased VE-cadherin expression and
stimulated Ep5ExTu cells to re-express E-cadherin at the cell surface. This promoted localization of -catenin at
the plasma membrane and induced the cells to undergo MET. Efficient silencing of N-cadherin expression in
Ep5ExTu cells consistently inhibited tumor growth, and complete tumor regression was even seen in some
cases. Taken together, these results reveal a novel interplay between classical cadherins in breast cancer
progression.
Discussion
Breast cancer is one the leading causes of death due to cancer worldwide. Although the genetic defects
underlying breast carcinogenesis have been extensively studied, important signaling pathways involved in the
progression of this specific tumor type are still poorly characterized. The loss of E-cadherin and concomitant
gain of N-cadherin expression is known to promote EMT and carcinoma progression. Our previous observation
that endothelial cell-selective VE-cadherin is expressed aberrantly in breast cancer cells and promotes their
proliferation both in vitro and in vivo [7] led us to analyze the specific roles of these cadherins as well as their
interplay in experimental breast cancer in more detail. Here, we show that N-cadherin silencing in murine breast
cancer cells suppresses tumor growth by upregulating E-cadherin, repressing EMT regulators, and reversing the
invasive mesenchymal phenotype to epithelial phenotype. Although both N-cadherin and VE-cadherin promote
tumor growth, their influence on E-cadherin expression in mesenchymal tumor cells is divergent: whereas N-
cadherin is capable of repressing E-cadherin expression in Ep5ExTu cells, VE-cadherin has no effect on its
expression levels.[7] Moreover, N-cadherin is required for maintaining VE-cadherin expression, but not vice
versa. The regulation of VE-cadherin expression by N-cadherin is a novel mechanism of tumor progression in
breast cancer and shows that N-cadherin both inhibits the expression of E-cadherin and stimulates the
expression of VE-cadherin.
The downregulation of VE-cadherin in the N-cadherin-deficient Ep5ExTu cells shows that N-cadherin is
required (although not necessarily sufficient) for VE-cadherin expression in aggressive carcinoma cells.
Regulation of VE-cadherin by N-cadherin was already described before in (nonmalignant) human umbilical
vein endothelial cells (HUVEC),[40] however, evidence for direct regulation of VE-cadherin by N-cadherin is
lacking, and the precise mechanisms involved in this regulation remain to be determined. The ability of N-
cadherin to regulate VE-cadherin was nonreciprocal because VE-cadherin silencing had no effect on N-cadherin
expression. However, as described also for other cell types,[28,29] VE-cadherin expression in Ep5ExTu cells
affected the localization of N-cadherin protein. In control Ep5ExTu cells, which express both cadherins, N-
cadherin displayed a nonjunctional distribution whereas in Sh-VE-cadherin knockdown cell lines, N-cadherin
was enriched at cell-cell junctions. Whether VE-cadherin expression can influence signaling pathways regulated
by N-cadherin as a consequence of excluding it from cell contacts remains to be determined.
There is growing evidence indicating that EMT is a reversible process in cancer cells. Recently, it was
hypothesized that tumor cells in metastatic sites can undergo re-differentiation and undergo MET.[1,3,6] This
transition could allow metastatic cells to adapt to a new microenvironment. Re-expression of E-cadherin is a
critical component of the MET.[41,42] However, little is known about the exact mechanism and biological or
clinical significance of MET in cancer. Islam et al. reported that blocking N-cadherin expression upregulates E-
cadherin expression in squamous epithelial cells.[10] Interestingly, we observed that N-cadherin silencing
promoted multiple aspects of MET in Ep5ExTu cells in a concentration-dependent manner, including
morphological changes, increased levels of E-cadherin and decreased levels of mesenchymal markers. In
contrast, VE-cadherin silencing led only to a weaker induction of epithelial markers and had no effect on E-
cadherin expression, indicating that MET is activated more efficiently by N-cadherin silencing than by VE-
cadherin silencing. This difference might be explained by the difference in -catenin localization in Sh-N-
cadherin and Sh-VE-cadherin cell lines. Interestingly, in Sh-N-cadherin cell lines (Sh-Ncad1.1 and Sh-Ncad2)
that displayed the most efficient N-cadherin downregulation, -catenin was localized at the cell membrane like
in the epithelial Ep5 cell line. As reported by other groups, alteration of -catenin localization alone can be
sufficient for the suppression of an invasive phenotype.[24,43]
The intermediate filament vimentin is an important marker of EMT and its expression is related to the adhesion
and migration properties of tumor cells.[44] A previous study using human breast cancer cells showed that
accumulation of cytoplasmic or nuclear -catenin and vimentin expression coincide.[36] Additionally, -catenin
can directly transactivate vimentin expression through its binding to the T cell factor (TCF)/lymphoid enhancer
factor (LEF) 1 transcription factor family. Vimentin expression was consistently downregulated in mammary
carcinoma cell lines in which -catenin was localized at the plasma membrane (Sh-Ncad1.1 and Sh-Ncad2), but
vimentin levels remained unchanged in lines that that showed cytoplasmic and/or nuclear distribution of -
catenin (Sh-Ncad1.2 and Sh-VEcad1).
Several transcriptional regulators are known to repress E-cadherin expression and thereby induce EMT. Among
these, we analyzed the expression level of Snail and SIP1, which emerged as key factors regulating E-cadherin
expression.[45] Whereas the level of Snail was downregulated in all Sh-N-cadherin cell lines, the level of SIP1
was decreased only in the Sh-Ncad1.1 and Sh-Ncad2 cell lines, which expressed higher E-cadherin levels. This
result therefore suggests that the re-expression of E-cadherin is stimulated more efficiently if the expression of
both transcriptional repressors of E-cadherin is decreased.
Deregulation of E-cadherin in breast cancer correlates with higher tumor grade and metastatic tumor cell
behavior.[46,47] Also in other cell types and in animal models, E-cadherin has been shown to act as a suppressor of
tumor growth and invasion.[48] In our study, suppressing N-cadherin significantly reduced Ep5ExTu tumor
growth. Remarkably, Sh-Ncad1.1 and Sh-Ncad2 cell lines hardly grew in vivo, and mice injected with either cell
line were often tumor-free 14 days after inoculation. Histological analysis of tumor sections isolated at day 10
post injection confirmed the re-expression of E-cadherin and downregulation of vimentin in Sh-Ncad2 tumors
in vivo. Since N-cadherin silencing did not change the proliferation rate of Sh-Ncad1.1 and Sh-Ncad2 in vitro, it
is likely that the phenotypic reversion of these cell lines, along with E-cadherin expression and associated -
catenin, leads to the inhibition of tumor growth in vivo. In contrast, moderate suppression of N-cadherin in Sh-
Ncad1.2, which greatly inhibits VE-cadherin expression, resulted in a growth rate similar to the Sh-VEcad2 cell
line. This growth inhibition correlates well with the decrease in cell proliferation observed for both cell lines in
vitro. It is therefore possible that the growth inhibition of the Sh-Ncad1.2 cell line is caused primarily by the
strong VE-cadherin suppression.
Does the introduction of VE-cadherin in N-cadherin (and consequently VE-cadherin) deficient cells restore
tumor growth? The forced expression of VE-cadherin in the Sh-Ncad2 cells (that had undergone MET) did not
change their epithelial phenotype, as indicated by unchanged E-cadherin and vimentin expression levels.
Additionally, junctional localization of E-cadherin was preserved in VE-cadherin re-expressing cell lines. In line
with the unaltered epithelial phenotype, forced expression of VE-cadherin failed to evoke a significant
difference in tumor growth; the VE-cadherin-expressing and control cells had similar growth rates in vivo.
These results suggest that VE-cadherin expression, at least in the presence of E-cadherin, is not sufficient to
promote tumor progression.
Conclusions
Our study shows for the first time that N-cadherin and VE-cadherin are co-expressed in human breast cancer. N-
cadherin controls the expression of VE-cadherin in aggressive mouse breast cancer cells and has an important
role in maintaining the mesenchymal phenotype and promoting tumor progression. VE-cadherin, on the other
hand, regulates the subcellular localization of N-cadherin by displacing it from the cell surface. Our study
supports the hypothesis that the interplay between cadherins in breast cancer progression is not limited to the
classical 'cadherin switch', which involves the loss of E-cadherin expression or function and the gain of N-
cadherin, but comprises also an intricate interdependence of N-cadherin and VE-cadherin.
References
Oncogene (2013) 32, 13161329; doi:10.1038/onc.2012.138; published online 30 April 2012
Glucocorticoids and histone deacetylase inhibitors cooperate to block the invasiveness of
basal-like breast cancer cells through novel mechanisms
M E Law1,2, P E Corsino1,2, S C Jahn1,2, B J Davis1,2, S Chen3, B Patel2,4, K Pham1,2, J Lu2,4, B Sheppard5,
P Nrgaard6, J Hong7, P Higgins8, J-S Kim1,2,9, H Luesch2,10 and B K Law1,2
Aggressive cancers often express E-cadherin in cytoplasmic vesicles rather than on the plasma membrane and
this may contribute to the invasive phenotype of these tumors. Therapeutic strategies are not currently available
that restore the anti-invasive function of E-cadherin in cancers. MDA-MB-231 cells are a frequently used model
of invasive triple-negative breast cancer, and these cells express low levels of E-cadherin that is mislocalized to
cytoplasmic vesicles. MDA-MB-231 cell lines stably expressing wild-type E-cadherin or E-cadherin fused to
glutathione S-transferase or green fluorescent protein were used as experimental systems to probe the
mechanisms responsible for cytoplasmic E-cadherin localization in invasive cancers. Although E-cadherin
expression partly reduced cell invasion in vitro, E-cadherin was largely localized to the cytoplasm and did not
block the invasiveness of the corresponding orthotopic xenograft tumors. Further studies indicated that the
glucocorticoid dexamethasone and the highly potent class I histone deacetylase (HDAC) inhibitor largazole
cooperated to induce E-cadherin localization to the plasma membrane in triple-negative breast cancers, and to
suppress cellular invasion in vitro. Dexamethasone blocked the production of the cleaved form of the CDCP1
(that is, CUB domain-containing protein 1) protein (cCDCP1) previously implicated in the pro-invasive
activities of CDCP1 by upregulating the serine protease inhibitor plasminogen activator inhibitor-1. E-cadherin
preferentially associated with cCDCP1 compared with the full-length form. In contrast, largazole did not
influence CDCP1 cleavage, but increased the association of E-cadherin with -catenin. This effect on E-
cadherin/-catenin complexes was shared with the nonisoform selective HDAC inhibitors trichostatin A (TSA)
and vorinostat (suberoylanilide hydroxamic acid, SAHA), although largazole upregulated endogenous E-
cadherin levels more strongly than TSA. These results demonstrate that glucocorticoids and HDAC inhibitors,
both of which are currently in clinical use, cooperate to suppress the invasiveness of breast cancer cells through
novel, complementary mechanisms that converge on E-cadherin.

The Biology of Breast Cancer


Lisa A. Carey, MD CME Released: 10/13/2010; Valid for credit through 10/13/2011

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.

Bone Drug Denosumab (Prolia) May Block Breast Tumors


LONDON (Reuters) Sep 30 - The same mechanism that makes Amgen's new bone drug denosumab (Prolia) an
effective treatment for osteoporosis may also mean it could help prevent breast cancer, scientists say.
Two papers published online September 29th in Nature report that the key osteoclast differentiation factor
RANKL, which helps regulate bone resorption, also has a role in breast cancers induced by endogenous
hormones.
The finding suggests that hormone-induced breast cancer could be blocked by denosumab, the first in a new
class of drugs that inhibit RANKL.
"Further studies will be needed to prove the principle of our findings," said Dr. Daniel Schramek, who worked
on the research with Dr. Josef Penninger at the Institute of Molecular Biotechnology in Vienna. "But we hope
that medical trials using denosumab can be started in the near future to test whether the mouse studies can be
directly translated to human breast cancer."
Dr. Schramek and Dr. Penninger and their colleagues found that treating mice with medroxyprogesterone
acetate (MPA) triggers massive induction of RANKL - which stands for "receptor activator of nuclear factor
(NF)-kappa B ligand" -- in mammary-gland epithelial cells. On the other hand, inactivation of the RANKL
receptor in these cells prevented them from proliferating and increased their rate of apoptosis.
Furthermore, deleting the RANKL receptor from the mammary epithelial cells "results in a markedly decreased
incidence and delayed onset of MPA-driven mammary cancer," the research team reports.
In a second study in Nature, also using mice, scientists at the U.S. biotech giant Amgen found that found that
RANKL is responsible for the major proliferative response of mouse mammary epithelium to progesterone
during mammary lactational morphogenesis. Induced activation of RANKL causes the mammary cells to divide
and multiply and fail to die when they should, and the inappropriate proliferation ultimately leads to breast
cancer, according to the report.
"The permissive contribution of progesterone to increased mammary cancer incidence is due to RANKL-
dependent proliferative changes in the mammary epithelium," the authors report.
Blocking the RANKL mechanism not only reduced breast tumor formation but also decreased the spread of
cancers to the lungs.
Prolia won approval from European regulators in May and from U.S. regulators in June for treatment of the
brittle bone disease. It is now under priority review by the U.S. Food and Drug Administration as a treatment for
patients with advanced cancers that have spread to the bones.
Nature. Posted online September 29, 2010. Abstract, Abstract

Epigenetics in Breast Cancer


What's New?
Yi Huang; Shweta Nayak; Rachel Jankowitz; Nancy E Davidson; Steffi Oesterreich
Posted: 03/23/2012; Breast Cancer Research. 2011;13(6):225 2011 BioMed Central, Ltd.
Abstract and Introduction
Abstract
Epigenetic changes are critical for development and progression of cancers, including breast cancer. Significant
progress has been made in the basic understanding of how various epigenetic changes such as DNA
methylation, histone modification, miRNA expression, and higher order chromatin structure affect gene
expression. The present review will focus on methylation and demethylation of histones. While the acetylation
of histones has been at the forefront of well-characterized post-translational modifications of histones, including
the development of inhibitors targeting de-acetylating enzymes, the past few years have witnessed a dramatic
increase in knowledge regarding the role of histone methylation/demethylation. This is an exciting and rapidly
evolving area of research, with much promise for potential clinical intervention in several cancers including
breast cancer. We also summarize efforts to identity DNA methylation signatures that could be prognostic
and/or predictive markers in breast cancer, focusing on recent studies using genome-wide approaches. Finally,
we briefly review the efforts made by both the National Institutes of Health Epigenome Project and The Cancer
Genome Atlas, especially highlighting the study of breast cancer epigenetics, exciting technological advances,
potential roadblocks, and future directions.
Introduction
While the term epigenetics is often used loosely, and sometimes in rather different ways, the term is generally
considered to encompass changes in DNA methylation, histone modifications, miRNA expression, and nucleo-
some positioning and higher order chromatin as epigenetic changes affecting gene regulation. Epigenetics was
defined as a discipline more than 50 years ago, by CH Waddington, and originally described changes in the
development of organisms that could not be explained by changes in DNA. Subsequently it became clear that
epigenetic modifications play important roles in diseases, including breast cancer. There is thus a pressing need
to understand the functional genome; that is, the changes defined by regulatory mechanisms overlaying the
genetic structure.
Over the past few years there has been an explosion in studies of epigenetics in breast cancer, reflected by the
exponential increase of published manuscripts (Figure 1). A PubMed search for the keywords 'epigenetic' and
'breast cancer' reveals that the first publication was in 1983. Progress was slow until approximately 10 years ago
when the number of studies started to steadily increase, at least in part fueled by improved technologies. In the
present review, we focus on recent advances in the understanding of histone methylation and demethylation, a
relatively new area with promise for clinical translation. We also review recent studies that have utilized
genome-wide technologies for the study of DNA methylation. Much progress has been made in the
characterization of noncoding RNAs, and the effect of higher order chromatin structure on gene expression in
breast cancer; however, these discoveries lie outside the scope of our review.
Figure 1. Increased rate of publication in the area of epigenetics and breast cancer. Data are derived from
a PubMed citation analysis searching for 'breast cancer' and 'epigenetics', and are approximate reflections of the
number of epigenetic studies in the breast cancer area.
Finally, we also discuss the relatively slow translation of results from the epigenetic field into the clinic.
Although there has been a dramatic increase of research into the epigenetics of breast cancer and milestone
discoveries have undoubtedly been made, the application of such findings into the clinical setting has been slow.
This is in contrast to other areas - for example, profiling of gene expression, where we have witnessed a
revolution in the past 4 to 6 years, especially in the translation of the results into the development of US Food
and Drug Administration-approved multigene prognostic assays. Why have we not yet seen any
predictive/prognostic tests that involve the characterization of epigenetic changes? In a similar way, although a
number of drugs targeting epigenetic changes have been tested, at this time no epigenetic drug has received US
Food and Drug Administration approval in breast cancer treatment. Is this a result from a slower development of
techniques used for epigenetic analysis? Or are there additional obstacles? In the present review article we
discuss some barriers to more rapid translation of epigenetic studies in breast tumors into clinical practice, and
discuss the efforts by the Epigenome Project and The Cancer Genome Atlas (TCGA) that are expected to bring
dramatic progress in the near future.
Acetylation and Methylation of Histones in Breast Cancer
For many years it has been known that post-translational modifications of histone tails determine, in part, which
regions of the genome are in an open and thus transcriptionally active conformation, and which are closed and
thus transcriptionally inactive. The modifications of histone tails include acetylation, methylation,
ubiquitylation, phosphorylation, sumoylation, and ribosylation, each of which can significantly affect the
expression of genes.[1] The most studied histone modifications are histone acetylation/deacetylation, and more
recently methylation/demethylation. In breast cancer, abnormal histone modification in combination with DNA
hypermethylation is frequently associated with epigenetic silencing of tumor suppressor genes and genomic
instability.[2,3] Understanding the mechanisms of dysregulation of histone tail post-translational modifications
and their contribution to breast tumorigenesis is critically important in the development of novel targeted
therapy for breast cancer patients.
Inhibition of Histone Deacetylases as a Therapeutic Approach in Breast Cancer
The dynamic nature of histone acetylation is determined by the counterbalancing activity of histone
acetyltransferases and histone deacetylases (HDACs). The HDAC family is divided into zinc-dependent
enzymes (classes I, IIa, IIb, and IV, of which there are 11 subtype enzymes) and zinc-independent enzymes
(class III, also called sirtuins), which require NAD+ for their catalytic activity.[4] Over the past decade, a number
of HDAC inhibitors have been rationally designed and synthesized based on their chemical structures and
divided into four groups: hydroxamic acids, cyclic tetrapeptides, short-chain fatty acids, and benzamides.[5]
Most of the HDAC inhibitors developed so far are nonselective, and among the most potent inhibitors are those
that have been designed to target primarily the zinc cofactor at the active site of the HDACs and to exhibit their
effects in the nanomolar or micromolar range.[6,7] Some of these HDAC inhibitors were shown to change the
chromatin structure and cause re-expression of aberrantly silenced genes, which in turn is associated with
growth inhibition and apoptosis in cancer cells.[8,9] In estrogen receptor (ER)-negative breast cancer cells,
inhibition of HDAC activity by specific HDAC inhibitors reactivates ER and progesterone receptor (PR) gene
expression, which are known to be aberrantly silenced.[1014] Pruitt and colleagues demonstrated that inhibition
of class III HDAC SIRT1 using a pharmacologic inhibitor, splitomicin, or siRNA reactivates epigenetically
silenced SFRP1, SFRP2, E-cadherin, and CRBP1 genes in human breast cancer cells.[15]
The study of HDAC inhibitors is moving rapidly into a new stage of development that has now started to
produce encouraging results in the clinic, particularly in the field of cancer therapy. Vorinostat (SAHA) and
romidepsin (FK228) have already been approved by the US Food and Drug Administration for the clinical
treatment of cutaneous T-cell lymphoma. Vorinostat is currently under evaluation in several phase II trials in
breast cancer,[16,17] including combination therapy of vorinostat with standard cytotoxic agents (for example,
paclitaxel), endocrine therapy (tamoxifen), or novel targeted therapy (trastuzumab, bevacizumab).[3,16,17] Other
HDAC inhibitors such as MS-275 (entinostat) and LBH-589 (panobinostat) are in phase I/II studies in
combination with other agents, such as trastuzumab, in women with metastatic HER2-positive breast cancer.[16]
In addition, increasing evidence suggests that combination treatment with inhibitors of HDAC and DNA
methyltransferase (DNMT) results in synergy at clinically tolerable doses that may translate not only into
changes in methylation but also to disease response. Preclinical studies of HDAC inhibitors in combination with
DNMT inhibitors have shown superior re-expression of silenced genes and increased apoptosis in colon/lung
cancer cell lines,[18] reduced tumorigenesis in lung cancer models,[19,20] superior ER re-expression compared with
HDAC inhibitor alone in breast cancer cell lines,[12] and restoration of tamoxifen responsiveness.[13,21] In a phase
I clinical trial of phenylbutyrate in combination with the DNMT inhibitor 5-azacitidine in myelodysplasia,
response was highly correlated with reversal of aberrantly methylated genes.[22] In another phase I trial in
nonsmall-cell lung cancer, the combination of a DNMT inhibitor and an HDAC inhibitor was safe and tolerable,
and was associated with clinical activity.[23]
An ongoing phase II trial is testing the HDAC inhibitor entinostat (also known as SNDX-275 and MS-275), in
combination with 5-azacitidine, in patients with hormonerefractory or triple-negative metastatic breast cancer.
The primary endpoint will be the objective response rate; secondary endpoints will be progression-free survival,
overall survival, and clinical benefit rate, as well as safety and tolerability. Other analyses will include the
pharmacokinetics of 5-azacitidine and entinostat, cytidine deaminase activity, pharmacogenetics, and baseline
and change in gene methylation in circulating DNA prior to/following combination therapy (quantitative
multiplex methylation specific PCR). The study will also aim to evaluate baseline and change in malignant
tissue via mandatory biopsies prior to/following combination therapy of gene methylation of candidate genes
(by quantitative methylation specific PCR) and of genomewide methylome, coupled with the study of candidate
gene re-expression (RT-PCR). We are at a critical turning point, because results from these critical studies will
guide future trials with HDAC inhibitors.
Targeting Histone Lysine Methylation and Demethylation in Breast Cancer
Histone lysine methylation is a reversible process, dynamically regulated by both lysine methyltransferases and
demethylases (Figure 2). In general, methylation of histone H3 lysine 4 (H3K4me), H3K36, or H3K79 is
associated with active transcription, whereas methylation of H3K9, H3K27, or H4K20 is associated with gene
silencing.[1] Histone methylation is regulated in breast cancer in an even more complicated manner than histone
acetylation via a large number of chromosomal remodeling regulatory complexes.
Figure 2. Model of dynamic interplay of enzymes mediating methylation of histone lysines. Methylases
are shown in pink and demethylases are shown in brown.
Modification of H3K4 methylation is catalyzed by the Trithorax group of histone methyltransferases, including
SET1 and MLL.[24] The activity of Trithorax proteins is balanced by the opposing effects of the Polycomb group
factors, another important histone methyltransferase family that mediates methylation usually associated with
epigenetic gene silencing.[25] Polycomb group proteins form at least four different complexes, including the
maintenance complex PRC1 - composed of RING, HPC, HPH, and BMI1 - and three different initiation
complexes, PRC2 through PRC4, which are formed by core component of zeste homolog 2 (EZH2), suppressor
of zeste 12 (SUZ12), and Nurf-55.[26,27] EZH2 is a highly conserved histone methyltransferase that specifically
targets H3K27 and functions as transcriptional repressor.[28] Tissue microarray analysis of breast cancers
identified consistent overexpression of EZH2, which was strongly associated with tumor aggressiveness.[29]
Studies from several groups demonstrated that expression of EZH2 is significantly associated with increased
proliferation and other features of aggressive breast cancer, such as p53 alterations, c-erbB-2 expression,
markers of the basal-like subtype, and glomeruloid microvascular proliferation.[30,31] Finally, in a recent report
by Chang and colleagues, EZH2 was shown to repress DNA repair in breast-tumor-initiating cells, potentially
leading to expansion of stem-cell-like cells, and finally to breast cancer progression.[32] Collectively, these
results suggest that EZH2 might function as a prognostic bio-marker in breast cancer, and might also be a
promising treatment target.
Histone lysine-specific demethylase 1 (LSD1, also known as BHC110, AOF2, or KDM1) is the first identified
histone lysine demethylase capable of specifically demethylating monoethylated and dimethylated lysine 4 of
histone H3 (H3K4me1 and H3K4me2).[33,34] The discovery of LSD1 has revolutionized the concept of histone
methylation as a dynamically regulated process under enzymatic control, rather than chromatin marks that could
only be changed by histone replacement. The activity of the LSD1-CoREST-HDAC complex has been
implicated in tumorigenesis. A recent study using ELISA determined that LSD1 is highly expressed in ER-
negative breast tumors, and hence LSD1 was suggested to serve as a predictive marker for aggressive breast
tumor biology and a novel attractive therapeutic target for treatment of ER-negative breast cancers.[35] In ER-
positive human breast cancer MCF-7 cells, 42% and 58% of all Pol II and ER-bound promoters, respectively,
were found to be bound by LSD1, and the recruitment of LSD1 to the promoters of LSD1+/ER+ target genes
was stimulated by estradiol.[36]
Intriguingly, Perillo and colleagues reported that LSD1-mediated demethylation produces H2O2, which
subsequently modifies the surrounding DNA and recruits 8-oxoguanine-DNA glycosylase 1 and topoisomerase
II, triggering conformational changes in DNA and chroma-tin that are essential for estrogen-induced
transcription.[37] Our recent study demonstrated that LSD1 interacts closely with HDACs in human breast cancer
cells. Importantly, inhibitors of histone demethylation and deacetylation exhibit cooperation and synergy in
regulating gene expression and growth inhibition, and may represent a promising and novel approach for
epigenetic therapy of breast cancer.[38] Recent studies also revealed that LSD1 is able to demethylate nonhistone
substrates such as p53 and DNMT1, indicating broader biological functions for LSD1.[39,40]
Subsequent to the discovery of LSD1, other Jumonji C (JmjC) domain-containing proteins were proposed to
function as human histone demethylases. These enzymes use -ketoglutarate and iron as cofactors to
demethylate histone lysine residues through a hydroxylation reaction.[4144] Little is known about the role of
JmjC domaincontaining histone demethylase in breast cancer, but recent studies found that PLU-1 (also known
as JARID1B or KDM5B) contributes to MCF-7 cell proliferation by facilitating G1 progression. Further,
knockdown of PLU-1 led to a significant reduction of MCF-7 cell proliferation and upregulation of expression
of certain tumor suppressor genes, including 14-3-3, BRCA1, CAV1, and HOXA5.[45] Sharma and colleagues
reported that the development of drug-tolerant cancer cells was at least in part mediated by activities of the
histone demethylase JARID1A/KDM5A. While these studies focused on epidermal growth factor receptor-
targeting small-molecule inhibitors in lung cancer cells, the authors showed that a similar mechanism for
resistance existed for other therapies, such as cis-platin. One could thus rationalize that activation of this
pathway might be a more widespread phenomenon for the development of drug resistance in
JARID1A/KDM5A-expressing tumors.[46,47]
Emerging Therapeutic Potential of Histone Methyltransferase and Demethylase Inhibitors
in Breast Cancer
As depicted in Figure 2, histone methylation is the result of a dynamic equilibrium between activities of a
number of histone methyltransferases and demethylases. Given the increasing evidence for their role in
tumorigenesis, it is no surprise they are being developed and tested as novel treatment targets.
Enhanced activity of histone-modifying enzymes such as LSD1 and EZH2 leads to epigenetic silencing of
critical genes, such as tumor suppressor genes, that have been shown to play an important role in breast tumor
tumorigenesis. A series of novel compounds function as powerful inhibitors of histone methylation or
demethylation and are capable of inducing re-expression of aberrantly silenced genes important in breast
tumorigenesis. A list of identified histone methyltransferase and demethylase inhibitors is presented in Table 1.
One of the first histone methyltransferase inhibitors developed is chaetocin, which exhibits some selectivity for
the SUV39 class of histone methyltransferases.[48] The EZH2 inhibitor DZNep induces robust apoptosis in
breast cancer cells, at least in part by including a novel apoptosis effector, FBXO32.[49] SMYD3 is a H3K4-
specific methyltransferase that is frequently overexpressed in a variety of cancers, including breast cancer.[50]
Novobiocin, known as a HSP90 inhibitor, decreases the expression of SMYD3 and inhibits the proliferation and
migration of MDA-MB-231 cells in a dose-dependent fashion.[51]
Table 1. Characteristics of some histone methyltransferase and demethylase inhibitors
The structural and catalytic similarities of LSD1 and monoamine oxidase or polyamine oxidase provided the
rationale to investigate whether existing monoamine oxidases or polyamine oxidase inhibitors might also act as
inhibitors of LSD1. Subsequently, the monoamine oxidase inhibitors tranylcypromine, clorgyline, and pargyline
were shown to inhibit LSD1 activity and inhibit growth of breast cancer and prostate cancer cells.[35,52,53]
Interestingly, pargyline (Eutonyl; Sigma-Aldrich, St Louis, MO, USA) has already been clinically used for the
treatment of vascular hypertension, and tranylcypromine (Parnate; Sigma-Aldrich, St Louis, MO, USA) is a
drug used as an antidepressant and anxiolytic agent in the clinical treatment of mood and anxiety disorders.
Unless there are toxicities due to the high doses that might be required to inhibit LSD1, one might expect this
drug to be tested in the cancer arena soon.
More recently, polyamine-based LSD1 inhibitors were identified and demonstrated to reactivate
epigeneticsilenced tumor suppressor genes in cancer cells.[54,55] Treatment with the LSD1-inhibiting polyamine
analogues 2d or PG-11144 significantly enhanced global H3K4me2 and altered gene expression in breast cancer
MDA-MB-231 cells.[56] Treatment with the LSD1 inhibitor PG-11144 and the DNMT inhibitor 5-aza-2-
deoxycytidine resulted in significant inhibition of the growth of established tumors in a xenograft model of
human colon cancer in nude mice.[55] N-oxalylglycine, an analog of -ketoglutarate, has been shown to be an
inhibitor of the JmjC domain-containing histone demethylases JMJD2A and JMJD2C.[57] These advances show
the promise of using novel compounds that target the histone methylation/demethylation pathway as an
innovative approach to breast cancer treatment, and are anticipated to lead to the development of a new
generation of therapeutically effective epigenetically-active drugs with considerable clinical potential.
The DNA Epigenome in Breast Cancer
Single-marker Studies in Breast Cancer
Over the past decade, significant progress has been made in the identification and characterization of altered
DNA methylation in breast cancer development and progression. A number of genes have been consistently
reported to be methylated, including RASSF1A, ER, PR, RAR, CCND2, and PITX2. We will not review
these findings here, but would like to point the interested reader towards a number of comprehensive reviews on
this topic.[20,58,59] The other frequently hypermethylated gene with a tumor-specific methylation profile is
BRCA1.[60,61] Although it has become clear that inactivation of BRCA1 by epigenetic means is a critical event in
breast (and ovarian) tumorigenesis, differences in experimental approaches and also in the region of the BRCA1
promoter analyzed resulted in ranges of methylation, and thus warrant some further analysis. In any case, in the
present review we will focus on results from some recent genome-wide methylation studies in breast cancer.
Analysis of the Breast Cancer Epigenome Using Genome-wide Approaches
Unprecedented advances have been made in the development of techniques to study genome-wide DNA
methylation. Briefly, there are currently four major approaches to identify 5-methylcytosine: restriction
endonucleasebased analysis, bisulfite-conversion of DNA, affinity and immunoprecipitation-based studies
(methyl Cp6 binding domain (MBD) pulldown, or antibodies against 5-methylcytosine in DNA or against
proteins binding to 5-methylcytosine, such as MBD2 and MeCP2), and finally mass spectrometry-based
analysis. Most of the approaches have been adapted to be used for genomewide studies using array-based or
sequencing-based methods, and some have been utilized to study methylation of the breast cancer genome, as
discussed below.
In 2007 Ordway and colleagues used cytosine methylation-dependent restriction enzyme McrBC coupled with
array hybridization to analyze methylation in nine matched invasive ductal carcinoma and adjacent normal
tissue.[62] They identified 220 differentially methylated loci, and analyzed 16 genes that were able to
differentiate breast tumor from normal and benign tissues and blood in more detail. One of these genes was
GHSR, a member of the G-protein-coupled receptor that binds to ghrelin. Methylation of GHSR was able to
differentiate invasive ductal carcinoma from normal or benign breast tissue with high specificity and sensitivity.
The same group went on to study four of the highly methylated loci - GHSR, max gene-associated, nuclear
factor I/X, and an unannotated region on chromosome 7 - in more detail through bisulfite pyrosequencing using
DNA from breast tumors, normal breast, and sera from cancer patients and from normal controls.
Disappointingly, no tumor-specific methylation pattern could be identified, and high methylation rates were
detected in normal sera.[63] The latter would clearly pose a problem for the development of sera-based assays
and highlights the need to identify markers that are not methylated in normal serum.
Ruike and colleagues reported results from a genome-wide methylated DNA immunoprecipitation sequencing
study in breast cancer cells.[64] Briefly, they identified methylated DNA in eight breast cancer cell lines and one
normal breast cell line, and in addition they compared methylation rates between parental MCF-7 cells and
MCF-7 cells that had undergone epithelial to mesenchymal transition. As expected, the cancer cell lines were
characterized by global hypomethylation, concurrent with hypermethylation of many loci. The
hypomethylation, which was distributed throughout the entire genome, was three to five times more frequent
than hypermethylation, which was clustered at specific loci. Intriguingly, 53% of methylated CpG was found
outside CpG islands. Of interest was also the association of epithelial to mesenchymal transition with
hypomethylation at many CpG islands, a finding that deserves follow-up.
It will be of great value to apply these approaches to answer clinical questions, such as the association of
genome-wide changes in DNA methylation with different grades or stages of breast tumor. A recent genome-
wide study by Fang and colleagues[65] has suggested that a CpG island methylator phenotype (CIMP) exists in
breast cancer. This breast cancer CIMP provided a distinct epigenomic profile, which was associated with genes
that make up the metastasis transcriptome. Additional studies need to be performed before one can confidently
state that there are CIMP tumors associated with specific tumor phenotypes. Another interesting question is the
association between methylation and molecular subtypes of breast tumors.
A recent study by Holm and colleagues suggests that luminal tumors have higher frequencies of methylation
compared with basal or triple-negative breast tumors.[58] Briefly, the authors studied 189 frozen primary breast
tumors using the Illumina Golden Gate Methylation Cancer Panel, covering 1,505 CpG loci in 807 cancer-
related genes. Unsupervised clustering revealed that methylation patterns were associated with luminal A,
luminal B, and basal-like tumors, with luminal B tumors being most methylated and basal-like tumors being the
least methylated. As previously reported, Her-2 tumors are very heterogeneous and are mainly driven by
amplification of Her-2 as the common denominator. High expression of PRC2 and low methylation of known
PRC2 targets in basal-like tumors suggest that PRC2 targets might be silenced by trimethylation of H3K27 in
this tumor subset. In general, these data clearly suggest that methylation plays a significant role in the different
breast tumor subsets, and it will be critical to determine the mechanism that drives different methylation states.
The authors speculate a role for genetic changes in methylation enzymes, an interesting hypothesis that is
testable.
Lineage-specific methylation was also observed in a methylation study performed by Sproul and colleagues,[66]
who used 27K Infinium arrays to study the methylation at > 14,000 genes in 19 breast cancer cell lines and 47
primary tumors. The authors bring forward the argument that DNA methylation in breast tumors is a marker of
cell lineage rather than tumor progression. This study again emphasizes the need to identify tumor-specific
methylation events, a task most critical for the future use of DNA methylation for diagnosis and treatment of
breast cancer.
Another clinical question is the involvement of DNA methylation in the adaptation of cancer cells to treatment
exposure. We recently performed an MBD-pulldown assay in breast cancer cells deprived of estrogen, thus
mimicking treatment with aromatase inhibitors. This approach resulted in the identification of a large number of
hypermethylated genes, and fewer that were hypomethylated (Pathiraja and colleagues, manuscript in
preparation). It will be of great interest to expand those studies to clinical samples, in order to identify markers
of resistance, and potential drug targets.
Clearly, these studies are only the beginning for the use of genome-wide methylation studies. With the advent of
improved technologies, we should expect to witness an explosion of studies aimed at understanding epigenetic
changes in breast cancer. This not only refers to DNA methylation, but also to other epigenetic changes, such as
histone modification, which can now also be studied genome wide through the use of chromatin
immunoprecipitation technologies At least in part, these efforts should soon benefit from the results of the
Human Epigenome Project and TCGA, as briefly discussed below.
Epigenome Project and TCGA: Their Role in Understanding Epigenetics of Breast Cancer (and
Other Diseases)
Description of Epigenome Roadmap Initiatives
The Epigenome Project is a Roadmap initiative led by several National Institutes of Health (NIH) centers,
started in 2008 when the NIH decided to invest over $190 million to accelerate the advancement of biomedical
research in epigenomics. A series of five initiatives was therefore created. The first initiative is the creation of
reference epigenome mapping centers, which support the development of reference epigenomes of a variety of
human cells, including normal breast epithelial cells. The data gathered include information on DNA
methylation, histone modifications, and associated noncoding RNAs. The second initiative focuses not only on
coordinating the banking of data, but also on facilitating its access to the public, accomplished through the
creation of the Epigenomic Data Analysis and Coordination center. The third initiative seeks to advance
technology in epigenetic research, by enabling the development of new techniques, including the creation of
methods that allow in vivo imaging of epigenetic changes. The objective of the fourth initiative is to identify
epigenetic marks and establish their function in mammalian cells. Finally, the intention of the fifth initiative is
to identify those epigenetic changes that are the cause of specific diseases, including breast cancer.
Importantly, the NIH roadmap initiative is part of an international association, the International Human
Epigenome Consortium, which has made several recommendations with regard to data release, format, and
various technical considerations, in order to universalize and validate findings.[67] Regarding the former, it is
recommended that all data be made available through one of several public databases, such as GEO,
ARRAYEXPRESS, and DDBJ.
Funded roadmap initiatives have resulted in many fundamental contributions, including a study published late
in 2009 that presented the first genome-wide, singlebase resolution map of methylated cytosines in the
mammalian genome from both human embryonic stem cells and fetal fibroblasts.[68] Importantly, almost
onequarter of all methylation identified in stem cells was in a non-CpG context, a finding that does not seem to
be restricted to methylation in stem cells. Subsequently, an approach was developed to sequence chromatin-
immunoprecipitated DNA from limited cell populations, an approach most critical for working with clinical
samples.[69] A study by Ernst and Kellis described a multivariate Hidden Markov Model to reveal chromatin
states in human T cells through the systematic analysis of 51 chromatin states, including promoter-associated
states, transcription-associated states, active intergenic states, large-scale repressed states, and repeat-associated
states.[70]
Finally, two studies have explored the strengths and weaknesses of four methods of DNA methylation mapping
technologies, while providing recommendations on the design of case-control studies in epigenomics.[71,72] These
studies mark a critical milestone for the Human Epigenome Project, since the development of genome-wide
technology has been a major focus on the initiative. Briefly, six methods were tested, of which five were
sequence-based and one was array-based. Each method was subjected to rigorous testing, and to statistical
analysis of at least two replicate samples. Although resolution and coverage differed, there was high
concordance between the different methods, providing a high level of confidence for all epigenetic researchers,
and providing flexibility as to which methods to choose based on the need for resolution, the amount of
available starting material, and, last but not least, the budget.
NIH Roadmap Studies Deciphering the Breast Cancer Epigenome
The initiative also funded disease-specific studies, including those in breast cancer. One such study is the
analysis of special AT-rich sequence binding 1 (SATB1) in metastatic breast cancer. The Kohwi-Shigematsu
laboratory identified SATB1, originally described as a genome organizer in thymocytes,[7375] to be a key
determinant in breast cancer metastasis.[76]
While there is some controversy about the detailed function of SATB1 in breast cancer,[77] there is no doubt that
SATB1 functions as a critical, global, genome organizer by recognizing and binding to specialized DNA
sequences in the genome that have a high propensity to unwind (base-unpairing regions). SATB1 organizes
chromatin into loops through binding of base-unpairing regions, which are found in gene-rich regions, and can
regulate a large number of distally located genes by functioning as a landing platform for multiple chromatin
remodeling/modifying proteins that confer specific epigenetic marks.[78] In breast cancer, once SATB1 becomes
expressed, it regulates ~1,000 genes, including those involved in cancer progression, metastasis, and growth (for
example, ERBB2, transforming growth factor beta).
The Kohwi-Shigematsu group is currently using genome-wide approaches to map all base-unpairing regions in
the genome and to determine which particular subset of these is bound by SATB1, and whether these specific
epigenomic modifications are associated with poor-prognosis expression profiles of aggressive breast cancers.
In addition, using a new approach of analyzing three-dimensional gene interactions, they have found that the c-
MYC gene locus is frequently brought into close proximity with a multitude of genes related to myc or co-
amplified in cancer. Forced SATB1 expression in nonaggressive breast cancer cells led to a major change in c-
MYC interaction pattern, establishing new connections with genes, some of which are related to cancer (T
Kohwi-Shigematsu, personal communication). This study provides a concrete example of how the Epigenome
Project supports the understanding of the progression from nonaggressive breast cancer to metastatic cancer by
establishing genome-wide changes in epigenetic marks, at least in part through global reorganization of higher
order chromatin structures by proteins such as SATB1 and others. The critical role for chromatin-organizing
proteins is reflected by frequent mutations, such as the recently described mutation of ARID1A in ovarian
cancer[47] and other cancers.[79]
In addition, beginning in October 2010, the Epigenome Project began to release data that included more than
300 maps of epigenetic changes in over 56 cell and tissue types, including a number of normal breast cells.[80]
For example, one can access data on DNA methylation as well as a number of critical genome-wide histone
markers (for example, methylation at lysines K4, K9, K27, and K36). These data should help to define breast
cancerspecific changes, by allowing researchers to compare the breast cancer data with the normal reference
epigenome. This, however, brings up one of several hurdles the project must overcome. There are challenges
regarding data integration, interpretation, and dissemination - as one would expect given that the technologies
used are all relatively new. There is a critical need for the creation of a new generation of tools for interpretation
of the numerous epigenetic datasets.[81] Briefly, in contrast to DNA sequence data, epigenomic data are not
digital, differ in resolution, and are highly variable. These features make comparisons of epigenomes
challenging, and require sophisticated informatics tools often not easily accessible for just any general
laboratory. Increasing accumulation of data, coupled with improved data and tool integration, and access to
computing resources and services, preferably through well-established and proven pipelines, are necessary for
the efficient and successful analysis of the unprecedented increase of epigenetic information.
Epigenetic Studies in Breast Cancer as Part of the Cancer Genome Atlas
TCGA began in 2006 as a combined effort by the National Cancer Institute and the National Human Genome
Research Institute. The success of the three-year pilot project led the NIH to commit major resources to TCGA
to collect and characterize more than 20 tumor types, including breast cancer. Tumor DNA and RNA will be
thoroughly characterized using a number of approaches. Data are currently available for the brain tumor
glioblastoma multiforme (GBM) and ovarian cancer, and we can expect more completion of the breast cancer
studies by the end of this year.
Currently, epigenomics studies within TCGA use the HumanMethylation27 BeadChip (Illumina, San Diego,
CA, USA). This assay allows quantitative interrogation of 27,578 CpG loci covering more than 14,000 genes at
single-nucleotide resolution. Specifically, the panel targets CpG sites located within the proximal promoter
regions of 14,475 consensus coding sequences, and in 110 miRNA promoters. As of April 2011 these data have
been available from the TCGA data portal for more than 400 breast cancers,[82] and we can look forward to the
report of its first analysis. Of note, a similar TCGA-directed approach in GBM resulted in the identification of a
unique glioma CIMP in about 10% of patients, who are usually very young at the time of diagnosis. These
patients survive more than 3 years, which is in stark contrast to most GBM patients who survive fewer than 15
months. Interestingly, the study also revealed an association between glioma CIMP with an acquired mutation in
the IDH1 gene.
The development of these technologies is moving very rapidly, and just when we thought we had a battery of
gold standards for genome-wide analysis of DNA methylation it becomes clear that additional modifications
such as hydroxymethyl cytosines, and methylated cytosines outside mCpG islands and outside promoter
regions, are likely to play critical roles, including in breast cancer. One may have speculated that the sole
analysis of promoter methylation - as done in the TCGA studies - might miss cancer-specific changes in other
critical regulatory regions. The exciting findings from the GBM study, however, clearly show that promoter
methylation includes clinically significant information, and we should look forward to additional data and
analyses from the breast cancer TCGA studies.
Conclusions
While an understanding of epigenetic changes in breast cancer has yet to be translated into clinical care, we
should expect major steps forward over the next few years. Fundamental discoveries in the understanding of
basic epigenetic regulatory mechanisms and dramatic advances in powerful technologies, together with large
national and international epigenome projects, will enable identification of breast cancer-specific alterations,
and thus potential predictive markers and treatment targets. We firmly believe we have entered an era of
epigenomics that will bring benefits for breast cancer patients.
References
Does Tumor Size Trump Biology?
Sunil Badve, MD, FRCPath
Posted: 04/23/2010; Clin Breast Cancer. 2010;10(2):111-112.
Introduction
In early days of chemotherapy, tumors > 2 cm were considered high risk and merited the use of adjuvant chemotherapy. More
recently, in the 1990s, when taxanes were introduced, the trials were open for patients with node-positive or high-risk node-
negative breast cancer. The definition of high risk was > 1 cm irrespective of estrogen receptor (ER) status. The decisions with
regard to size and biology were also made easier by the fact that there is a good correlation between tumor size and grade; larger
tumors are generally of high grade. As we understand more and more about tumor biology and as the tumor sizes continue to
plummet, the question of relative importance of these distinct parameters of assessing tumors has come to the forefront.
In this issue of the Clinical Breast Cancer, Chreau et al present the data with regard to the outcomes of breast cancers detected
by magnetic resonance imaging (MRI) screening in patients with BRCA1 or BRCA2 mutations.[1] They compare the outcomes
with a similar group of patients diagnosed by more traditional means. As one might expect, they find that patients diagnosed
through the use of MRI had smaller tumors. However, this did not translate into significant differences in 3-year disease-free or
overall survival. They state that the treatment decisions were made as per the St. Gallen guidelines. [2] For patients hormone
receptor (HR)negative or high-histologic-grade tumors with node-negative disease, these guidelines recommend the use of
chemotherapy only if the tumors are > 1 cm. Because significant numbers of the patients with BRCA mutations have high-grade
HR-negative tumors but were under 1 cm in size, these patients did not get adjuvant chemotherapy. In fact, patients in the group
diagnosed through MRI were half as likely to receive adjuvant chemotherapy (43% vs. 86%).
Several studies have analyzed the issue of prognosis in T1ab N0 breast cancers. Leitner et al analyzed a series of 218 patients
and found that poor nuclear grade and presence of lymphatic invasion identified a small subset of patients with significant risk
of recurrence that warranted adjuvant systemic therapy.[3] Studies from the Finland and British Columbia Cancer registries have
documented recurrence rates between 15% and 30% for these small tumors. [4,5] More recently, there have been back-to-back
articles on the outcomes in small HER2-positive tumors, irrespective of their ER status. [68] These show that HER2 positivity is
associated with a two- to five-fold increase in the absolute risks of recurrence. In addition, there is some evidence to suggest
that these patients are as likely to benefit from anti-HER2 therapies as those patients with larger tumors. [9]
There is a dearth of studies analyzing the prognosis of small tumors in the triple-negative population. Evans et al, in a study of
1944 women with screen-detected tumors < 15 mm, showed that basal phenotype was significantly associated with poor
survival.[10] In addition, there are several aspects of triple-negative tumors that are unique. Size does not appear to be a reliable
prognostic factor in these tumors.[11] These tumors tend to spread via the hematogenous route rather than by classical lymphatic
route;[12] thus, the tumors are more likely to be understaged.
All of these articles bring home a simple message: ignore biology at your peril. So the next question that needs to be answered
is how low does one go?
The past often provides clues about the future. Since the early 1970s, evaluation of HR status, in addition to size, is routinely
performed in the management of invasive cancers. The St. Gallen guidelines recommend the use of chemotherapy for patients
with ER-positive tumors only for large ( T2) tumors.[2] Perhaps the treatment algorithm for the treatment of small (T1ab N0)
tumors might be similar to that used in the management of ductal carcinoma in situ, where multiple parameters are used to guide
therapy. It is quite possible that a multi-parametric equation that takes into account the patient parameters such as age and
menopausal status; tumor characteristics such as histology, immunophenotype, and size; as well as type of surgery and width of
margins will enable educated decision making for these small tumors. Based on the recent data in ER-positive tumors, [13] one
may also postulate that race could play a role in determining outcome.

Hes-6, an Inhibitor of Hes-1, is Regulated by 17-estradiol and Promotes


Breast Cancer Cell Proliferation
Johan Hartman; Eric W-F Lam; Jan-ke Gustafsson; Anders Strm
Posted: 03/31/2010; Breast Cancer Research. 2009;11(5):R79
Abstract
Introduction Hes-6 is a member of the basic helix-loop-helix (bHLH) family of transcription factors, and its overexpression has been
reported in metastatic cancers of different origins. Hes-6 has been described as an inhibitor of Hes-1 during neuronal development,
although its function in cancer is not known. In this study, we investigated the function of Hes-6 in breast cancer and tested the
hypothesis that Hes-6 enhances breast cancer cell proliferation and is regulated by estrogen.
Methods To investigate the function of Hes-6, T47D cells stably expressing Hes-6 were generated by lentiviral transduction, and
conversely, siRNA also was used to knock down Hes-6 expression in breast cancer cells. The Hes-6-expressing T47D cells were
transplanted into immunodeficient mice to study effects on tumor growth.
Results We found that Hes-6 expression was significantly higher in the high-grade, estrogen receptor (ER)-negative SKBR3 and
MDA-MB-231 cells compared with the ER-positive, non-metastasizing T47D and MCF-7 breast carcinoma cells. Moreover, the
level of Hes-6 mRNA was 28 times higher in breast cancer samples compared with normal breast samples. In Hes-6-expressing T47D
cells, Hes-6 ectopic expression was shown to stimulate cell proliferation in vitro as well as breast tumor growth in xenografts.
Moreover, expression of Hes-6 resulted in induction of E2F-1, a crucial target gene for the transcriptional repressor Hes-1.
Consistently, silencing of Hes-6 by siRNA resulted in downregulation of E2F-1 expression, whereas estrogen treatment caused
induction of Hes-6 and downstream targets hASH-1 and E2F-1 in MCF-7 cells.
Conclusions Together, the data suggest that Hes-6 is a potential oncogene overexpressed in breast cancer, with a tumor-promoting and
proliferative function. Furthermore, Hes-6 is a novel estrogen-regulated gene in breast cancer cells. An understanding of the role and
regulation of Hes-6 could provide insights into estrogen signaling and endocrine resistance in breast cancer and, hence, could be
important for the development of novel anticancer drugs.
Introduction
The majority of breast cancer cells are dependent on estrogens to support their survival and proliferation. [1] 17-Estradiol (E2) is the
most potent estrogen as well as the predominant estrogen in premenopausal women. In breast cancer, two main types of estrogen
receptors (ERs) exist, ER and ER.[24] As shown by in vitro experiments, ER mediates the proliferative effect of estrogens, whereas
ER inhibits proliferation[5] in breast cancer cells. In T47D and MCF-7 breast cancer cells, ER promotes proliferation by stimulating
expression of cell-cycle regulators and through downregulation of the transcriptional repressors, such as Hes-1. Hes-1 is a member of
the basic helix-loop-helix (bHLH) family of transcription factors, [6] first described in embryonic development, in which Hes-1 inhibits
differentiation of developing neurons. In breast cancer cells, downregulation of Hes-1 is essential for estrogen-mediated proliferation.
[7]
Consistently, forced expression of Hes-1 causes G1-phase cell-cycle arrest. The transcriptional activator E2F-1 is an important cell-
cycle regulator, stimulating the G1/S-phase transition by activating the transcription of other cell-cycle genes. [8] We earlier identified
E2F-1 as a crucial transcription factor directly inhibited by Hes-1 at the transcriptional level in breast cancer.[9] Hes-1 binds to the
promoter region of E2F-1, thereby repressing its transcription. Based on our findings, we believe that E2F-1 is a central factor in Hes-
1-mediated inhibition of proliferation.
Hes-6 is a member of the same family of transcription factors as Hes-1 but functions as a posttranslational inhibitor of Hes-1. [10,11] Hes-
6 forms a heterodimer with Hes-1, thereby preventing its association with transcriptional co-repressors. Hes-6 was first discovered in
nervous tissue, but its expression in the mammary gland is not known. Despite its role as an inhibitor of Hes-1, the function of this
potential oncogene remains unclear.
Human achaete-scute complex homologue 1 (hASH1) is another member of the bHLH-family. In contrast to Hes-1, hASH-1 functions
as a transcriptional activator, inducing transcription through E-boxes, and is negatively regulated by Hes-1 at the promoter level. [12,13]
Despite being a potential tumor suppressor in vitro, no significant difference in its expression between breast cancer and normal tissue
has been found. Therefore, another cofactor is probably involved in the regulation of Hes-1 action.
In an experimental mouse model of colon cancer, several genes were upregulated in metastases, but the only gene that was
upregulated in all metastases compared with their primary tumor was Hes-6. Furthermore, the authors showed that Hes-6 is
upregulated in several types of human cancers compared with normal tissue.[14] Recently, Hes-6 and hASH-1 have been reported to be
overexpressed in high-grade prostate cancer and were suggested to be involved in neuroendocrine development of the cancer cells to
an aggressive phenotype.[15]
By expressing Hes-6 in the breast cancer cell-line T47D, we studied its role in tumor growth and proliferation. In addition, we
investigated its effects on expression of the Hes-1 target gene E2F-1 and its potential involvement in ER signaling. Because Hes-6
antagonizes Hes-1, our hypothesis is that Hes-6 increases the proliferation of breast cancer cells and is regulated by estrogen.
Discussion
We previously identified and characterized Hes-1 as an essential transcription factor in ER-signaling and a strong inhibitor of
estrogen-stimulated proliferation in breast cancer cells. [7] However, despite several attempts to investigate Hes-1 expression in breast
cancer, we have not found any difference in Hes-1 expression in breast cancer tissues compared with normal tissues. For this reason,
we investigated the expression of the Hes-1 inhibitor Hes-6 and its potential involvement in proliferation.
As described in the Introduction, the transcription factor Hes-6 is a novel Hes-family member, whose only established function is to
inhibit DNA binding of Hes-1, thereby inhibiting Hes-1 activity. Here we identify Hes-6 as a novel estrogen-regulated gene in breast
cancer. Most interestingly, as shown in Figure 1, Hes-6 is expressed at higher levels in breast cancer tissue compared with normal
breast tissue. This result could be interpreted in at least two different ways: Hes-6 could be a marker associated with breast cancer in
general, without any major function. Alternatively, it could mean that Hes-6 is induced in early stages of breast cancer and is important
for the progression of breast cancer. In this study, we present evidence that Hes-6 has an important role in the proliferation of breast
cancer cells and in the growth of corresponding xenografts.
As expected, in parallel to the high levels of Hes-6, we identified a higher expression of E2F-1 in the breast cancer samples compared
with that in the normal breast tissue samples (Figure 1c), in agreement with what has been described by other researchers. Based on
our findings, we suggest that increased E2F-1 levels could be a result of regulation by the Hes-1/Hes-6 system.
As shown in Figure 1a, Hes-6 mRNA also was higher in the aggressive, ER-negative cell lines SKBR3 and MDA-MB231 than in the
ER-positive T47D and MCF-7 cells. Because Hes-6 has been identified as a marker for aggressive, high-grade cancers of other
origins than the breast, Hes-6 also could be associated with high proliferation and aggressiveness in breast cancer. Conversely, this
finding is in contrast to our experiments implicating Hes-6 as a gene expressed in response to ER stimulation (Figure 4). It is likely
that other, ER-independent signaling pathways also regulate Hes-6 expression. This is exemplified by the nerve growth factor activity
of Hes-1 during differentiation of the pheochromocytoma cell line PC12.[18] Because genetic abnormalities are common in breast
cancer,[19] it also is possible that the DNA regions harboring the Hes-6 gene are amplified in some breast cancer cells, thereby causing
constantly increased Hes-6 levels.
The function of Hes-6 has been studied extensively within the developing nervous system. [10,11] Moreover, the expression of Hes-6 in
different tissues has been reported. Nevertheless, this is the first study in which the function of Hes-6 in cancer is described.
As shown in Figure 3, expression of Hes-6 caused increased proliferation in both the absence and the presence of E2, The most
probable explanation is that Hes-6 inhibits Hes-1, thereby changing the expression of cell-cycle regulators. E2F-1 might be
particularly important in this context, because it is strongly inhibited by Hes-1 at the transcriptional level. [9] In agreement with this
notion, expression of Hes-6 resulted in induction of E2F-1 at both the mRNA and the protein levels, whereas inhibition of Hes-6 by
siRNA prevented the ER-mediated induction of E2F-1 (Figures 2 and 5).
In the xenograft experiments shown in Figure 3, the expression of Hes-6 resulted in a dramatic increase of tumor growth, most likely
as a consequence of the proliferation-stimulatory function of Hes-6. However, expression of Hes-6 might also affect xenograft growth
in proliferation-independent pathways; for example, Hes-6 might regulate angiogenesis and paracrine growth factors. When Hes-6
was expressed in T47D cells, the levels of both Hes-6 and E2F-1 was higher in the cells grown as xenografts than in in vitro cultured
cells (Figures 2 and 3). A possible reason for this is that the three-dimensional in vivo milieu and the presence of stromal factors
stimulate Hes-6 expression further. We believe that alternative pathways can at least partially overcome ER regulation in subtypes of
breast cancer.
The important role of E2F-factors in cancer has been investigated by other researchers in great detail.[8,20] E2F-1 has been studied as a
potential marker in breast cancer diagnostics. In one report, E2F-1 protein correlated with Mib1/Ki67 expression and was expressed at
higher levels in advanced-stage breast cancer.[21] High expression of E2F-1 also has been shown in small cell lung cancer, and these
tumors do not express Hes-1. Conversely, non-small cell lung cancer expresses lower levels of E2F-1 and higher levels of Hes-1,
indicating that Hes factors may be important in E2F-1 regulation in these cancers as well.[22]
As shown in Figure 4, E2 treatment of MCF-7 cells caused increased expression of Hes-6 mRNA and protein. Because MCF-7 cells
contain only ER, this effect should be mediated through ER. However, transient transfections of T47D and MCF-7 cells with a Hes-
6 promoter construct did not reveal any ER-mediated regulation (data not shown). Accordingly, we speculate that an element
upstream or downstream of the proximal Hes-6 promoter is responsible for ER-regulated Hes-6 transcription.
In addition to Hes-6, we found that E2 treatment of MCF-7 cells resulted in increased hASH-1 expression. Moreover, hASH-1 was
expressed in response to Hes-6 in T47D cells.
Because hASH-1 is normally repressed by Hes-1 through an element in its proximal promoter,[23] it is likely that the induction of
hASH-1 is a consequence of Hes-6 expression, leading to inactivation of Hes-1. In tissues, Hes-6 and hASH-1 expression are often
associated with each other. For instance, it was recently shown that Hes-6 and hASH-1 correlate with more-aggressive prostate cancer.
[15]

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]

Aspirin May Boost Survival in Breast Cancer


February 17, 2010 The regular use of aspirin might increase survival in breast cancer, according to data published online February 16 in
the Journal of Clinical Oncology. Aspirin use was associated with a decreased risk for distant recurrence, breast cancer death, and death from
any cause.
Depending on the number of days of aspirin use per week, patients were able to reduce the risk for distant metastasis by 43% to 60%.
Likewise, compared with nonusers, aspirin use was associated with a 64% to 71% reduction in the risk for breast cancer-related mortality.
"It is important to realize that this is an observational study, and in such a study we cannot prove that aspirin causes improved survival," said
lead author Michelle D. Holmes, MD, DrPH, assistant professor of medicine at Harvard Medical School in Boston, Massachusetts. "It will be
important to see if our results can be repeated in other observational studies."
It will also be important, Dr. Holmes told Medscape Oncology, to undertake studies to determine how aspirin might improve survival.
The aspirin they took was not a substitute for these therapies.
She also emphasized that all women in this study had undergone conventional breast cancer treatment, including surgery, radiation,
chemotherapy, and hormonal therapy. "The aspirin they took was not a substitute for these therapies, it was on top of these therapies," she
said. "It is also important to understand that aspirin intake can have detrimental side effects, such as gastrointestinal bleeding."
To prove that aspirin can improve survival in women with breast cancer and to ultimately change clinical practice would require a clinical
trial, Dr. Holmes added. "However, women with breast cancer who are already advised by their doctors to take aspirin for another reason
might take comfort in the fact that the aspirin might also be preventing recurrence of their breast cancer."
Previous Studies Inconclusive
Experimental studies have shown that aspirin can inhibit growth and decrease the invasiveness of breast cancer cells, reduce cytokines
involved in bony metastasis, and stimulate immune responsiveness, the authors write. In addition, meta-analyses of aspirin or other
nonsteroidal anti-inflammatory drugs have shown a 9% to 30% reduced risk for breast cancer incidence associated with their use. However,
the authors note that the association between aspirin use and breast cancer incidence remains inconclusive.
In the current study, the authors hypothesized that aspirin use after a diagnosis of breast cancer would be associated with both a lower risk for
breast-cancer-related death and distant recurrence among women with stage I to III disease.
They conducted a prospective observational study involving 4164 female participants in the Nurses' Health Study (NHS) who were
diagnosed with stages I, II, or III breast cancer between 1976 and 2002. The women were observed until June 2006 or their death, whichever
came first.
Within this cohort, there were 341 breast-cancer-related deaths, 400 distant recurrences (including the 341 breast cancer deaths), and 732
deaths from other causes. A total of 2910 women diagnosed with breast cancer did not provide an aspirin assessment after their diagnosis.
The primary outcome was breast cancer mortality risk according to the number of days per week of aspirin use (0, 1, 2 to 5, or 6 or 7 days),
which were assessed at least 12 months after their diagnosis and then updated accordingly.
We measured their aspirin intake after their breast cancer diagnosis.
"We measured their aspirin intake after their breast cancer diagnosis, being careful not to assess their aspirin intake in the first year after their
diagnosis when they might have been getting radiation and chemotherapy, when patients are advised not to take aspirin," said Dr. Holmes.
Their results showed that aspirin use was associated with a decreased risk for breast cancer death, and the results did not appreciably differ
when they were stratified by disease stage, body-mass index, menopausal status, or estrogen-receptor status. The results were all the "more
notable" because the NHS failed to find an association between aspirin use and breast cancer incidence, say the authors.
Compared with never users, the multivariate adjusted relative risks (RRs) for breast cancer mortality were:
0.88 (95% confidence interval [CI], 0.64 - 1.22) for past use
1.07 (95% CI, 0.70 - 1.63) for current use 1 day per week
0.29 (95% CI, 0.16- 0.52), for current use 2 to 5 days per week
0.36 (95% CI, 0.24 - 0.54) for current use 6 or 7 days per week.
The results were similar for distant recurrence; adjusted RRs were 0.91 (95% CI, 0.62 - 1.33), 0.40 (95% CI, 0.24 - 0.65), and 0.57 (95% CI,
0.39 - 0.82) for 1, 2 to 5, and 6 or 7 days of aspirin use, respectively.
In addition, aspirin use was associated with a decreased risk for mortality from any cause; multivariate RRs for overall mortality were 0.96
(95% CI, 0.76 - 1.21), 0.94 (95% CI, 0.67 - 1.32), 0.53 (95% CI, 0.37 - 0.76), and 0.54 (95% CI, 0.41- 0.70) for past, current 1 day per week,
current 2 to 5 days per week, and current 6 or 7 days per week of use, respectively.
For all-cause mortality, the authors note that the protective effect associated with aspirin appears to be "driven by the impact on breast cancer
death."
They speculate that the association between breast cancer death and aspirin use was stronger than with recurrence, "because recurrence is
more likely to be misclassified than death." There was also a "modest association" with the duration of aspirin use, and the authors note that
aspirin use "may influence proximal rather than distal events in the cancer pathway."
"If confirmed, our results may broaden the scope of interventions available to reduce breast-cancer-related morbidity and mortality," they
conclude.
The study was supported by a grant from the National Institutes of Health. The authors have disclosed no relevant financial relationships.
J Clin Oncol. Published online February 16, 2010.

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.

Metastatic Breast Cancer Survival Predicted by


Nodal Ki-67?
Diana Mahoney Nov 06, 2012
BOSTON The presence of high levels of the Ki-67 antigen in axillary lymph node metastases is a significant
and clinically useful predictor of survival in breast cancer and may identify subgroups of patients who would
benefit from more aggressive treatment, suggests a study presented here at the American Society for Clinical
Pathology (ASCP) 2012 Annual Meeting.
Assessment of the Ki-67 antigen has proven to be a simple and dependable method for indirectly measuring cell
proliferation, and studies have demonstrated a positive relationship between cell proliferation in breast cancer
and tumor grade and size, mitotic activity, hormonal and Her-2 status, and tumor progression, according to
Kareem Tawfik, MD, lead author and resident of the University of Cincinnati, Ohio.
In light of a growing body of evidence suggesting that proliferation markers may identify subgroups of patients
with breast cancer who are most likely to benefit from adjuvant chemotherapies, senior author Ossama Tawfik,
MD, PhD, director of anatomic and surgical pathology, Department of Pathology and Lab Medicine at the
University of Kansas Medical Center in Kansas City, Kansas, and colleagues compared the prognostic
significance of Ki-67 expression in axillary lymph node metastases to that of patients' matched primary tumors.
This research group previously reported a direct correlation between Ki-67 expression in breast cancer cells and
prognosis for survival.
In the current study, the investigators evaluated Ki-67 expression (by means of immunohistochemistry via the
MIB-1 monoclonal antibody) in the primary breast tumors and corresponding axillary lymph node metastases of
103 patients (median age, 54.5 years) treated at their institution and correlated the data with patient age, tumor
grade, tumor size, estrogen receptor, progesterone receptor, p53, epidermal growth factor receptor, Bcl-2, Her-2
status, and overall survival.
Of the 103 primary breast carcinomas, most of which (86%) were ductal type, 17 were Scarff-Bloom-
Richardson (SBR) grade I, 32 were SBR grade II, and 54 were SBR grade III, Dr. K. Tawfik said in a poster
presentation of the data. The median Ki-67 expression in primary and metastatic tumors, respectively, was 20%
and 15%, he said, noting that the latter was significantly lower for cases in which primary tumors were smaller
than 2.0 cm.
An analysis of the results showed no difference in overall survival between primary tumors with 10% Ki-67
expression or less, "but there was significantly better overall survival when Ki-67 expression in lymph nodes
was less than 10%," Dr. K. Tawfik reported. "For patients whose primary tumors exhibited Ki-67 expression
less than 10%, most of their metastatic lesions had a similarly low Ki-67, and they had a favorable outcome."
Worse survival was observed in the small subgroup of patients (6 of 34) with low Ki-67 expression in the
primary tumor who had a nodal Ki-67 of 10% or higher, he said.
Ki-67 in Lymph Nodes May Tell More Than in Primary Tumors
Of 69 patients whose primary tumors had high Ki-67 expression, most had similarly high nodal Ki-67
expression, with the exception of 12 patients in whom nodal Ki-67 expression was less than 10%. These 12
patients "had significantly better overall survival," Dr. K. Tawfik said, suggesting that the measurement of Ki-
67 in metastatic lymph nodes is more telling and potentially more clinically useful than its evaluation in
primary tumors. "Identifying subgroups of patients with different levels of Ki-67 expression in lymph nodes and
primary tumors may help in the selection of therapeutic options," he said. Specifically, "based on our findings,
patients with higher proliferative activity in lymph node metastases might require more aggressive therapy and
closer clinical monitoring of their disease."
Although Ki-67 is a robust and valuable biomarker of cell proliferation, the clinical utility of measuring its
expression is hampered at present by variations in scoring procedures and the lack of standardization for
different types of specimens, explains Mitch Dowsett, PhD, head of breast cancer translational research at the
Royal Marsden Hospital and Institute of Cancer Research in London, United Kingdom, and co-chair of the
International Ki-67 in Breast Cancer Working Group. "The direct application of specific cutoffs for decision
making must be considered unreliable" in the absence of intra- and interlaboratory standardization, he told
Medscape Medical News. Dr. Dowsett was not involved in this study.
Dr. K. Tawfik and Dr. Dowsett have disclosed no relevant financial relationships.
American Society for Clinical Pathology (ASCP) 2012 Annual Meeting. Poster 361. Presented November 2,
2012.

Molecular oncology: The positive in the negative


Kendall Powell Nature485,S52S53(31 May 2012)doi:10.1038/485S52aPublished online30 May 2012
Rushing between meetings at the University of North Carolina at Chapel Hill, oncologist Lisa Carey scoffs at
the current descriptor 'triple-negative breast cancer'. Defining anything by what it isn't is scientifically insane.
But, clinically, it's what we've got.
When patients are diagnosed with breast cancer, their tumours are classified into one of four pathological
subgroups based on whether the tumours express the oestrogen receptor (ER positive) or the progesterone
receptor (PR positive), overexpress one of the members of the epidermal-growth-factor receptor family (HER2
positive) or none of these a classification known as triple-negative breast cancer (TNBC). When cancers are
driven by the hormones or the HER2 pathway, the good news is that effective targeted therapies exist. For the
remaining cancers, there are no clear targets.
For patients with TNBC, conventional chemotherapy, which affects a wide range of dividing cells, is the only
available treatment after surgery and radiation. But this approach has met with mixed success. TNBC is often
aggressive and highly resistant to chemotherapy. The five-year survival rate for TNBC is about 77%, compared
with 93% for other types of breast cancer.
TNBC accounts for about 1020% of all breast cancer in the United States (see 'The hard facts', page S50), with
a higher incidence in certain populations. In African-American women, for example, it constitutes about 30% of
all breast cancers1. And in women who carry an inherited mutation in the BRCA1 gene, most breast-cancer cases
are TNBC. It also tends to be more common in younger women.
Mixed company
The news is not all bad. A fraction of TNBC cases respond well to chemotherapy given before surgery, with
some women even experiencing a complete response that is, no evidence of live tumour cells at the time of
surgery. And research into TNBC is still at an early stage specific clinical trials for TNBC did not begin until
2006. To make headway against TNBC, however, researchers must first come to grips with the burgeoning data
showing that this subgroup is heterogeneous.
Biologists are now beginning to divide TNBC into subcategories and to dissect the molecular signals driving
each one, with the hope of finding targets for clinical trials. We're acknowledging that this is not one disease,
and we're designing trials with that in mind, says Jennifer Pietenpol, director of the Vanderbilt-Ingram Cancer
Center in Nashville, Tennessee.
The trend of defining breast cancers by their molecular profiles (or gene-expression patterns) began in 2000,
with the publication of a landmark study2 led by Charles Perou, a cancer geneticist now at the University of
North Carolina, Chapel Hill. In the past decade, researchers have defined six molecular subtypes of breast
cancer, including two 'luminal' subtypes (which generally match ER-positive cancers) and a 'HER2-enriched'
subtype (which matches HER2-positive cancers).
A significant proportion of TNBC, 5075%, matches the molecular subtype known as 'basal-like' breast
cancers3. These are characterized by the high expression of genes that are normally expressed in the basal
epithelial layer of skin, such as the keratin-5, -6 and -17 genes. Most basal-like breast cancer is triple-negative;
however, not all cases are, so the two terms are not fully interchangeable.
If we are ever going to make therapeutic advancements in TNBC, we'll be targeting the 75% that is basal-like,
says Perou. He adds that anything that helps TNBC patients in a clinical trial must be working on this subset. I
always argue, let's track the biology and the dominant biology is the basal-like subtype.
Others favour different approaches, noting that focusing solely on this basal-like subtype of TNBC might not be
specific enough to yield broader therapeutic results. We should look at each tumour individually, says John
Carpten, a cancer genomicist at the Translational Genomics Research Institute in Phoenix, Arizona. Even
among basal tumours, growth can be driven by completely different molecular signals, so drilling down to the
actionable driving events for individual tumours seems like a rational approach as well.
Cell by cell
Carpten's group is 'deep sequencing' TNBC tumours, which involves sequencing multiple cells within a tumour
to identify coding mutations, deletions or translocations. His team will then merge this genomic information
with whole-transcriptome data (representing all the expressed genes). The hope is to eventually incorporate the
phosphoproteome: the set of proteins that is modified by addition of a phosphate group by protein kinases,
thereby altering the proteins' activity in intracellular signalling pathways. This approach, he says, will provide
doctors with a reference page for a patient's tumour that can point them to the drugs most likely to have the
biggest impact.
The first published deep-sequencing effort4 looked at 104 primary TNBC tumours and found a wide range of
genetic mutations. Some tumours had just a few coding mutations, whereas others had hundreds. This analysis,
led by molecular pathologist Samuel Aparicio at the BC Cancer Agency Research Centre in Vancouver, Canada,
showed predominant mutations in well-known tumour-suppressor genes, such as the gene encoding p53 (called
TP53), as well as in oncogenes. In several tumours, however, too few of the tumour cells contained these
mutations for them to have caused the cancer. Affirming Carpten's sentiments, Aparicio's group found that
basal-like TNBCs in fact showed more genetic variation at diagnosis than other TNBCs. There's no more
efficient way of learning that information about a tumour than sequencing it, says Aparicio.
The growth of ER-positive and HER2-positive cancers is largely driven by signalling through these receptors.
Now researchers are attempting to determine whether basal-like cancers or, importantly, other TNBCs also
share molecular drivers. We need to define these triple-negative cancers positively by their drivers, but we
aren't quite there yet, says Alan Ashworth, chief executive of the Institute of Cancer Research in London.
Until recently, the only candidates for defining TNBC were mutations in the BRCA1 gene, the tumour-
suppressor gene TP53 and, occasionally, mutations or loss of the tumour-suppressor gene retinoblastoma 1
(RB1). Even this limited information has been put to use, though.
In 2005, Ashworth and his colleagues discovered the first evidence that BRCA1 loss might be exploited for
TNBC therapy5. In a preclinical study, they described a combination of loss of BRCA1 and inhibition of the
enzyme poly(ADP-ribose) polymerase (or PARP) that was lethal to tumour cells. DNA damage, such as breaks
in the DNA, is caused by exposure to radiation, including sunlight and radiotherapy, and by the normal process
of cell division. PARP is involved in the repair of single-strand breaks in DNA, whereas BRCA1 participates in
double-stranded break repair. So BRCA1-deficient cells that also lack PARP activity cannot repair DNA damage
and subsequently die. This effect was so persuasive that by 2007 PARP inhibitors were being trialled in patients
carrying BRCA mutations.
The results of these trials have been mixed, however: BRCA1-mutation carriers responded well, but other TNBC
patients did not. (The trial results have also been confounded by the inclusion of one drug, iniparib, which is
unlikely to be a true PARP inhibitor.) There's a heterogeneity here that we don't understand, says Carey.
Maybe in TNBC that isn't driven by BRCA1, you have to kick DNA-damage repair in another way, perhaps by
hitting the tumour with direct DNA-damaging agents such as radiation or certain chemotherapeutics, causing
unrepairable breaks.
Another approach is being taken by Bryan Schneider and Milan Radovich, cancer researchers at Indiana
University in Indianapolis. They are looking for other weaknesses in TNBC cells by comparing the genomes
and transcriptomes of TNBC tissue with those of healthy breast tissue. As tumour cells are dividing very
rapidly, it was no surprise to find that PARP and genes in the BRCA1 pathway are upregulated in TNBC
samples. However, in as-yet unpublished research, Schneider and Radovich also discovered that a handful of
protein kinases involved in epithelial-cell development and cell survival were also expressed at high levels. We
were very surprised to find that the top overexpressed kinases have not been studied in breast cancer at all,
says Radovich a result suggesting new drug targets.
Classifying the unclassifiable
These investigations suffer from TNBC's heterogeneity, however. Mapping the genomic or transcriptomic
differences from normal tissue is likely to reveal the dominant features of basal-like TNBC. But there is
probably noise in these studies from the other, non-basal-like, TNBCs. Pietenpol's group has begun to tackle
that problem, essentially by subtyping the subgroup with striking results6.
Pietenpol and her team searched for gene-expression data for as many cases of TNBC as possible. The team
performed a cluster analysis on the resultant 587 tumours to identify gene-expression subtypes within TNBC.
This analysis found six distinct gene-expression profile clusters: basal-like 1 (BL1), basal-like 2 (BL2),
immunomodulatory, mesenchymal, mesenchymal stem-like and luminal androgen receptor (LAR). As expected,
the BL1 and BL2 subtypes dominated, making up about 50% of the TNBC samples. Surprisingly, however,
about 10% of women with TNBC those with the LAR subtype had tumours overexpressing the androgen
receptor, suggesting that these cancers are driven by hormones that are not usually associated with breast cancer.
Pietenpol suspects that androgen-receptor antagonists used for treating prostate cancer might be commandeered
for treating this TNBC subset. It's a wonderful example of how subtyping a disease at the bench gives pretty
quick insights as to how you could benefit patients, she says.
Pietenpol's group also showed that TNBC cell lines modelling these six subtypes responded to drugs that were
selected according to the gene-expression profile. For example, cells representing BL1 and BL2 subtypes are
predicted to have defective DNA repair and be genomically unstable. These cells were most sensitive to
cisplatin, a drug that induces DNA lesions and death in cells that can't repair DNA damage.
The future options for TNBC therapy are widening. Clinical trials blocking other molecular targets are under
way. Carey is involved in a trial of cetuximab, which inhibits the epidermal-growth-factor receptor (EGFR), a
molecular marker of basal-like tumours. So far, the gains with cetuximab have been modest, as tumours seem to
be finding ways to escape EGFR inhibition. Still, she's not giving up on this approach. These are among the
most genetically unstable tumours there are. Their ability to circumvent [treatments] is not that surprising.
And Pietenpol is keen to move discoveries to the clinic as quickly as possible. Patient numbers dictate that the
focus is on finding treatments for patients with basal-like breast cancer, but she is confident about opportunities
for the much smaller LAR patient group. Any progress made for any of the subtypes will be significant; it's no
different from Glivec, she says, referring to imatinib, the kinase-targeted drug that is highly effective against
several cancer subtypes. If you have a very good outcome based on a more precise therapy, then that can
change the face of oncology care for patients. And that's what we're in this for.

Metastasis: The rude awakening


Jocelyn Rice Nature485,S55S57(31 May 2012)doi:10.1038/485S55aPublished online30 May 2012
Ann Chambers has a series of graphs tacked up in her office at the London Regional Cancer Program in
Canada, where she is an oncologist. I stare at them all the time, she says.
These charts are assembled from more than 60 years of data gathered at the MD Anderson Cancer Center in
Houston, Texas, and show how the ten-year survival curves for local and metastatic breast cancer have changed
over the decades (see 'The hard facts', page S50). The picture they paint of breast cancer, Chambers says, has
regions of darkness and light.
On the one hand, the overall survival rate of patients with breast cancer has vastly improved. Sixty years ago,
only a quarter of patients were alive ten years after being diagnosed; now, that figure exceeds three-quarters. On
the other hand, for patients whose tumour has metastasized spread to distant sites outside the breast at the
time of diagnosis, the picture remains dismal. Even now, a patient with metastatic breast cancer has only a 22%
chance of surviving more than ten years.
The graph that troubles Chambers most, however, is the one for patients with local breast cancer who show no
evidence of metastasis at diagnosis. Decade after decade, the survival curves for these patient cohorts decline
over time, with the current ten-year survival rate at 86%. The implication is that, despite appearing free of
disease, there are hidden tumour cells in these women, says Chambers. These cells are lurking in a state of
suspended animation. It might be a rare event; however, even 25 years after diagnosis, these dormant cells can
reawaken, growing into a full-blown metastasis and ultimately killing a patient who was once considered to be
cured.
The cells that give rise to such late metastases remain mysterious. Where are they hiding? Why do they
reawaken? This is a very peculiar biology, says Klaus Pantel, an oncologist at the University Medical Center
Hamburg-Eppendorf in Germany. If we can understand what the body is doing to control this cancer for 10 or
15 years, and what stops this control and eventually leads to metastatic relapse, we can foresee completely new
strategies for controlling disseminated cancer.
Seeds and soil
One thing we do know about these dormant cells is where they originally came from. In most patients who
develop metastases, tumour cells have already undergone a cascade of transformations at the time of diagnosis,
allowing many of them to escape from the primary lesion (see 'The right trials', page S58). These cells enter the
bloodstream, either directly or by way of the lymph nodes, and circulate through the body. In cancer,
dissemination is an early event, says Christoph Klein, a cancer biologist at the University of Regensberg,
Germany. Indeed, research by Klein and others shows1, 2 that escaped tumour cells can sometimes be detected
even in patients diagnosed with ductal carcinoma in situ (DCIS), an early-stage breast cancer in which the
tumour appears to be confined to the lining of the milk ducts.
The escaped cells in the bloodstream, called circulating tumour cells (CTCs), are the potential seeds of future
metastases (see 'The great escape'). After a patient has completed chemotherapy, the presence of CTCs is
associated with a higher risk of recurrence. Monitoring CTCs during treatment might also provide a means to
track a patient's disease over time and adjust therapy accordingly, says Massimo Cristofanilli, a breast cancer
clinician and researcher at Fox Chase Cancer Center in Philadelphia, Pennsylvania. CTCs, he says, provide a
real-time assessment of the disease. For example, a patient might have a primary tumour that expresses a
normal amount of the receptor HER2 (a HER2-negative tumour). If later testing reveals CTCs that overexpress
HER2 (HER2-positive cells), this new information would expand treatment options making the patient a
candidate for HER2-targeted therapies, such as trastuzumab (Herceptin) and lapatinib. Before, you wouldn't
have even considered that, says Chambers. The expression of hormone receptors can also differ. Chambers
says that monitoring the oestrogen- and progesterone-receptor (ER and PR) status of CTCs could offer another
way to adapt a patient's treatment over time.
ADAPTED FROM AGUIRRE-GHISO ET AL. NATURE REVIEWS CANCER (2007)
CTCs are attractive candidates for prognosis and tracking disease status because they are accessible with a
simple blood draw. A device for detecting CTCs in blood has been approved by the US Food and Drug
Administration: CellSearch (produced by the diagnostics company Veridex, based in Raritan, New Jersey)
identifies tumour cells in a blood sample by using tiny magnetic particles coated with an antibody against a
known epithelial-cell marker. However, this marker is sometimes lost when cells leave the primary tumour, and
researchers estimate that CTCs are missed in up to one-third of patients. Alternative approaches use different
markers to distinguish the tumour cells or microfluidic technology to sort the larger tumour epithelial cells from
the smaller blood cells. Either way, it's a needle-in-a-haystack problem because white blood cells can
outnumber CTCs by 500,000 to 1, says David Lyden, a cancer biologist and paediatric oncologist at Weill
Cornell Medical College and Memorial Sloan-Kettering Cancer Center in New York.
Cellular re-education
Not all CTCs develop into a full-blown metastasis, says Chambers. They're merely seeds. To develop, they must
first find the appropriate soil. To some extent, she adds, the location of metastases is determined by the patterns
of blood flow that carry cells away from the primary tumour. After entering the bloodstream, tumour cells first
travel to the heart. From there, their next stop is the capillary beds of the lungs. Some cells may lodge there,
says Chambers, whereas others can weasel their way through, getting out into the circulation again to get
access to other sites.
Circulatory patterns don't tell the whole story, however. Certain tissues are especially hospitable to CTCs,
providing a microenvironment replete with the nutrients and growth factors that they need. Cells can get to all
sorts of places, says Chambers, but they'll grow where they find a good match with what they need. The best
'soil' for breast cancer cells, she says, can be found in the lungs, liver, brain and bones.
Complicating the seed-and-soil concept is the emerging realization that CTCs can send out signals free-
floating molecules such as growth factors and chemokines, as well as membrane-bound packets of protein and
nucleic acid called exosomes that can prime target sites to be more receptive to the cells' arrival. In other
words, the seed is fertilizing the soil in advance. Lyden and colleagues call these primed microenvironments
'pre-metastatic niches', and they suggest that metastases might be prevented by identifying and inhibiting these
priming signals.
The process of site remodelling continues after CTCs have established themselves in a new destination, where
they are now known as disseminated tumour cells (DTCs). Recent work suggests that this kind of
communication might also happen in reverse. Tumour cells that make it to the bones will encounter quiescent
stromal cells in the bone marrow. It's possible that tumour cells become educated by the surrounding cells,
says Lyden. In vitro studies have shown that in the bone-marrow stroma, microRNAs (short non-coding RNAs
with a regulatory function) can be taken up by breast cancer cells, inducing them to become quiescent3. Free-
floating microRNA from the bone marrow can re-educate tumour cells to be more like bone-marrow stem cells,
saying 'be quiet, don't proliferate, stay here in the bone marrow', says Lyden.
This process of re-education also raises another intriguing possibility, he says. Haematopoietic stem cells
(blood-cell precursors) can leave the marrow and circulate through the body. If DTCs have been reprogrammed
to behave like these cells, perhaps DTCs also take tours around the circulatory system. If they're in the bone
marrow, are they just sleeping there, asks Lyden, or are they like bone-marrow stem cells, which can go out
into circulation and come back? If the latter is true, the bone marrow could be a long-term reservoir for
metastatic disease. Patricia Steeg, a molecular biologist at the National Cancer Institute in Bethesda, Maryland,
highlights the finding that bone-marrow-derived cells are recruited to pre-metastatic niches elsewhere in the
body as evidence of this possibility. It certainly suggests that these bone-marrow cells could provide a safe
haven for dormant tumour cells, she says.
Pantel has been analysing DTCs in the bone marrow of patients with breast cancer for more than 20 years.
These cells are more difficult to study than CTCs, as aspirating bone marrow is a far more complicated and
painful procedure than drawing blood. But, like their counterparts in the blood, DTCs can offer crucial
information about a patient's prognosis and can appear in bone-marrow aspirates even when the primary tumour
is still very small. These cells are not just innocent bystanders, says Pantel. Their presence is predictive of a
poorer clinical outcome. Yet the fate of these cells is wildly variable. Some will grow into metastases
immediately; some will do so much later. And, he adds, if the cells do leave the bones for good, the
microenvironment at their ultimate destination might yet block their outgrowth, leaving them in a state of
lifelong dormancy.
This uncertainty is a problem for routinely testing patients for DTCs in the bone marrow, says Lyden. A
negative result is great news: the patient is very unlikely to develop bone metastases. But a positive result is
harder to interpret. What do you tell the patient? asks Lyden. It's impossible to know the fate of a given DTC.
When will it start proliferating? Will it proliferate at all? Researchers are searching for biomarkers (genetic or
biochemical signatures) that could hold the answers to such questions. HER2 could be one such marker: as is
the case for CTCs, tumour cells found in the bone marrow can have a different HER2 status from the primary
tumour, and HER2-positive DTCs are more likely to grow into metastases than HER2-negative DTCs. A
multicentre study is now under way in Germany to investigate whether monitoring the HER2 status of DTCs
can guide treatment strategies for patients with HER2-negative primary breast cancer.
Reading the signs
Some researchers think that bones are the most important site of dormancy and metastasis in breast cancer; a
liver metastasis, for example, might grow from a cell that first spent a decade asleep in the bone marrow. Others
think that liver metastases grow from CTCs that travelled directly to the liver and then became dormant.
Animal studies support the latter idea that dormancy is a systemic phenomenon. In one such study4, pathologist
David Tarin and colleagues at the University of California, San Diego, injected human breast cancer cells
tagged with green fluorescent protein into the mammary fat pads of mice. Not only did mammary tumours form
but, in autopsy studies using fluorescence microscopy, green pinpoints were also evident in various other
organs. When the fluorescent cells were retrieved, even from organs with no overt metastases, the individual
apparently dormant cells were able to generate metastases in new mice, indicating that the cells had retained
their metastatic potential.
In humans, however, researchers don't have the option of tagging tumour cells for easier imaging. Analysis of
liver and bone-marrow biopsies can hint at the presence of tumour-derived cells, but there is currently no
imaging technique sensitive enough to locate individual DTCs inside the body. George Sledge, an oncologist at
the Indiana University School of Medicine in Indianapolis, says: Radiologists speak about the one-millimetre
challenge the difficulty of imaging structures smaller than 1 mm. An isolated DTC falls well outside that
range. Even in autopsy studies, in which a pathologist can assess tissues directly, identifying a solitary DTC is a
tough problem.
Underlying the difficulty of locating dormant tumour cells is a more fundamental issue: there's still no
consensus on what 'dormancy' entails. I'm still stumped on the definition of dormancy, says Steeg. In one
interpretation, says Sledge, tumour cells are actively dividing, but a barrier prevents them from crossing the
threshold to overt metastasis. That barrier could be blood supply: the micrometastasis is starved of nutrients
until a genetic switch flips, allowing the cells to develop their own vasculature. Alternatively, it could be
immune surveillance: the cells cannot proliferate beyond a micrometastasis until they evolve a strategy to evade
the constant policing of the immune system.
In a view championed by Chambers, dormancy in DTCs might be a truly quiescent state a kind of long-term
hibernation, where the cells might not be dividing at all. The mechanisms regulating this type of dormancy are
unclear, but recent research by Julio Aguirre-Ghiso, an oncology researcher at the Mount Sinai School of
Medicine in New York, indicates one possible scheme. Within cells, there is a balance between two signalling
pathways: the growth-promoting ERK pathway, and the growth-restricting p38 pathway5. Some tumour cells
lose p38 signalling, escaping quiescence and spurring their characteristic unchecked growth. Aguirre-Ghiso and
his colleagues have proposed that once these cells leave the primary tumour, certain microenvironments might
disturb this balance: an environment that preserves ERK dominance might yield an immediate metastasis,
whereas one that promotes p38 could lead to dormancy6. Aguirre-Ghiso calls this the target organ scenario:
The cells arrive, and they respond to that microenvironment.
But Aguirre-Ghiso and colleagues found that the fate of disseminating cells might already have been at least
partly determined in the primary tumour. By examining experimental dormancy gene-expression signatures
associated with the ERK and p38 pathways, Aguirre-Ghiso's team was able to predict with reasonable accuracy
in patients whether an ER-positive primary tumour would produce early metastases or have a propensity for
long-term dormancy7. This quiescence-associated signature could be influencing the fate of the cells that left
the primary tumour, says Aguirre-Ghiso. That would provide a mechanism to explain why some patients have
an early recurrence and why some patients have a later recurrence after clinical dormancy, he says.
Wake-up call
The challenge of understanding dormancy pales, however, in comparison to uncovering why dormant DTCs
reawaken. Researchers have plenty of ideas but little hard evidence. Lyden speculates that age-related changes
in the microenvironment, such as a shifting ratio of myeloid and lymphoid cells or a decrease in physical space
for haematopoietic stem cells, might stimulate DTCs to resume their growth. Infection or trauma for
example, a broken bone might have the same effect, says Lyden. Chambers, meanwhile, sees a role for diet
and exercise in regulating the recurrence of breast cancer (see 'Powering up', page S62). Prospective trials are
needed to examine the effects of such lifestyle changes over decades, she says. But for now data are scarce.
One thing is clear though. If breast cancer survival rates are to continue to improve, we need to redefine breast
cancer. Pantel foresees a future in which breast cancer is treated like a chronic disease, such as diabetes. With
appropriate drugs and lifestyle modifications, he says, we could prevent recurrence by encouraging dormant
cells to remain dormant or by destroying them before they reawaken.
On another level, Klein says, we need a new pathology of systemic cancer because the characteristic of the
disease is that it changes. It's evolving. It's progressing. Right now, patients receive treatments that are tailored
on the basis of information about the primary tumour. Tests can identify not only hormone-receptor and HER2
status, one of the standard tumour classifications, but also subtle genetic signatures that predict early recurrence.
While we have lots of genomic assays that predict metastasis, most are better at predicting early recurrence
rather than late recurrence, says Sledge. We clearly need better gene or protein signatures that predict late
relapse and people are working on this. The search for better diagnostic and prognostic clues will probably
have to shift away from the primary tumour towards the circulating and disseminated cells. Metastasis is a
moving target, and researchers acknowledge that any effective treatment strategy will have to constantly
recalibrate its aim.

Environment and genetics: Making sense of the


noise
Anna Petherick Nature485,S64S65(31 May 2012)doi:10.1038/485S64aPublished online30 May 2012
Possibly the most famous cancer gene is called BRCA1, for breast cancer 1. It is expressed in breast tissue and
encodes a protein that fixes double-stranded breaks in DNA. When such breaks cannot be repaired, the same
protein triggers cell suicide, protecting the cell from replicating the mutated DNA. Mutations in BRCA1 that
prevent its expression or cripple the protein raise the risk of developing breast cancer. A woman with a faulty
BRCA1 gene has a 65% chance of developing breast cancer by the age of 70 (ref. 1).
No other genes with an equivalent level of breast cancer risk have been found. And as the search for candidate
genes considers ever more subtle effects, the study population that is needed to provide the statistical power to
spot these effects is growing correspondingly larger. Finding BRCA1 and BRCA2 (another tumour-suppressor
gene linked to breast cancer) required only a few families with a high incidence of breast cancer and a panel of
only a few hundred genetic markers2. But now the search for correlations between disease incidence and
genetics routinely encompasses tens of thousands of people and scans the full length of their genomes. These
genome-wide association studies (GWAS) have so far identified about 25 genetic loci linked to breast cancer,
implicating a variety of cellular mechanisms in the disease.
The missing inheritance
Using GWAS does not definitively answer the question of which genes raise the risk of cancer, however. The 25
or so single nucleotide polymorphisms (SNPs) that have been linked to breast cancer3 label sections of DNA
that are typically tens or hundreds of kilobases long, rather than specific genes. These 'haplotype blocks' might
each contain several genes, any of which could be causing the association. Only in a few cases have researchers
confirmed the specific gene responsible, such as the fibroblast growth-factor receptor 2 gene, which was
discovered by a team led by Kerstin Meyer at the Cancer Research UK Cambridge Research Institute.
But GWAS fall short in other ways as well. The loci found so far through genetic screening account for only 9
10% of breast cancers. Many breast cancers are caused by gene variants too rare to show up in these studies,
and even after considering genes that have strong effects (such as BRCA1 and BRCA2), 70% of breast cancer
cases are still unexplained4. Yet having a family history of breast cancer is a far stronger predictor than the
genetics alone imply.
Shared environmental details of family life might play a part, of course, although results from studies on
identical twins raised apart suggest that this is unlikely. Calculations based on these studies reveal that having a
mother or sister with breast cancer doubles a woman's risk of developing the disease, without the environment
playing any role5. So where is the missing heritability?
Several large cohort studies are trying to plug this gap in our knowledge. Douglas Easton, head of the Cancer
Research UK genetic epidemiology group at the University of Cambridge, was keenly involved in the hunt for
BRCA1 and BRCA2. He is now running a study called EMBRACE, which is beginning to make sense of the
way the genetic variants interact. Half of the 3,000 women enrolled in EMBRACE have either a faulty BRCA1
or BRCA2 gene, and half do not. Using GWAS has helped to clarify the risk conferred by a harmful mutation in
these genes. The risk associated with a faulty BRCA1 gene is modified by the presence of other SNPs that have
been independently linked to oestrogen-receptor (ER)-negative breast cancers, so called because these cells
underexpress oestrogen receptors. Meanwhile, BRCA2 risk is affected by a different set of variants, which
makes sense because you tend to get ER-positive disease with BRCA2, says Easton.
Such combinatorial data are being added to a computer program called Boadicea (named for a historical British
warrior queen), developed at the University of Cambridge, which calculates the odds of breast cancer on an
individual basis. With these new data, instead of only being able to tell a woman with a BRCA2 mutation that
she has a 50% chance of developing the disease by the time she is 70, we can identify women with a 70% or
more risk, and women with a risk below 30%, says Easton.
Another intriguing cohort study is Breakthrough Generations. Anthony Swerdlow, an epidemiologist at the
Institute of Cancer Research in London who is joint head of the study, hopes that the repeated, detailed
questionnaires and blood samples supplied by the study participants will help distinguish genuine molecular,
behavioural and environmental risk factors from factors that merely correlate with them (for a different
approach, see 'A traumatic environment'). Many factors on the list obesity, exercise, the age at which
women go through the menopause might act via relatively few hormones, he explains.
Box 1: A traumatic environment: Is psychological stress a risk factor?
Full box
Swerdlow suspects that lifelong exposure to changing levels of these hormones, in particular during critical
periods of female life history (such as menarche and menopause), might amplify the impact of the genes. The
snapshots that one-time questionnaires and blood samples provide are useful, but they don't really get at the
subtle shifts over a woman's lifetime that are likely to increase or decrease her chance of developing breast
cancer, he adds. The study has already discovered independent yet useful details of life history, including four
common genetic loci that help to predict the age of early menopause and thus when a woman's fertility is
likely to start declining, which is typically ten years earlier6.
The history of risk
Breakthrough Generations includes women as young as 16, but determining the role of different aspects of
female life history in breast cancer risk requires cohort studies that follow girls while they develop breasts.
Ethical considerations make these trials harder to run, but a few groups are trying.
The University of Cincinnati in Ohio, the University of California, San Francisco, and the Fox Chase Cancer
Center near Philadelphia, Pennsylvania, have each recruited groups of about 400 girls aged 6 to 8. They plan to
follow these cohorts to at least the age of 17, to help understand the environmental and genetic factors that lead
to the onset of puberty, because earlier menarche is a risk factor for breast cancer in adults. At least once a year,
these 1,239 girls will be assessed for exposure to environmental toxins, including phyto-oestrogens and other
chemicals known to disrupt hormone levels, their body mass index will be calculated, and other medical and
social details will be recorded. But such a small study has its limits. We're nowhere near being able to really
understand the environmental influence, says Meyer.

Breast Cancer Intratumor Genetic Heterogeneity


Causes and Implications
Charlotte KY Ng, Helen N Pemberton, Jorge S Reis-Filho
Oct 31, 2012
Expert Rev Anticancer Ther. 2012;12(8):1021-1032. 2012 Expert Reviews Ltd.
Abstract and Introduction
Abstract
There is burgeoning evidence to suggest that tumor evolution follows the laws of Darwinian evolution, whereby
individual tumor cell clones harbor private genetic aberrations in addition to the founder mutations, and that
these distinct populations of cancer cells interact in competitive and mutualistic manners. The combined effect
of genetic and epigenetic instability, and differential selective pressures according to the microenvironment and
therapeutic interventions, create many different evolutionary routes such that intratumor heterogeneity is
inevitable. Numerous cytogenetic, comparative genomic hybridization and, more recently, massively parallel
sequencing studies have generated indisputable evidence of this phenomenon. The impact of intratumor
heterogeneity on response and resistance to therapy is beginning to be understood; this information may prove
crucial for the potentials of personalized medicine to be realized. In this review, the evidence of intratumor
heterogeneity in breast cancer, its potential causes and implications for the clinical management of breast cancer
patients are discussed.
Introduction
By the time of diagnosis, breast cancers are composed of heterogeneous populations of tumor cells. It is
becoming increasingly evident that intratumor genetic heterogeneity is one of the underlying causes of
resistance to systemic therapies. The prevailing hypothesis for the generation of intratumor heterogeneity,
emerging from massively parallel sequencing studies, is the clonal evolution model (Figure 1A). The clonal
evolution model postulates that tumors, with rare exceptions, originate from a single cell, and evolve through
the acquisition and selection of randomly generated genetic aberrations. [1] When most of the mutations are
likely to be deleterious or neutral under a given set of selective pressures and will likely become extinct, those
that confer selective advantage may lead to clonal expansion. However, many epithelial cancers, harbor up to
tens of thousands of somatic genetic aberrations at diagnosis, [25] suggesting that there may be many mutations
that are neither selectively advantageous nor deleterious at a given timepoint, the so-called 'passenger'
mutations. These 'passenger' mutations, albeit not conferring direct advantage to cancer cells under a given
repertoire of selective pressures, may contribute to intratumor phenotypic and genetic heterogeneity. [6] As the
selective pressure changes, for instance, between the microenvironment of the primary and metastatic site or
due to systemic therapies being administered, a different set of mutations may become advantageous (i.e., a
'passenger' mutation acquires properties of a 'driver' mutation). The presence of differential selective pressure
may, therefore, cause subclones to diverge, generating further intratumor heterogeneity. [6] It should also be
noted that in addition to genetic alterations, epigenetic events are also heritable and are also subject to selection.
[1]
Interestingly, massively parallel sequencing research endeavors have recently led to the identification of
somatic mutations affecting chromatin remodeling genes, which play pivotal roles in the regulation of
epigenetic phenomena. [7,8] These recent findings suggest that in some cancers, the dysfunctional epigenetic
regulation documented in cancers may ultimately be determined by the repertoire of genetic aberrations.

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.

Insights into Breast Cancer Heterogeneity


Posted: 05/31/2010; Nat Rev Cancer. 2010;10(3):163 2010 Nature Publishing Group
Introduction
Increasing evidence indicates that breast tumours are sustained by a population of cancer stem cells (CSCs). In a
recent study published in Cell, researchers led by Pier Paolo Di Fiore report the purification and molecular
characterization of normal human mammary stem cells (hNMSCs) from cultured mammospheres, and provide
evidence supporting a model in which breast tumour heterogeneity is a reflection of the number of CSC-like
cells in the tumour.
To isolate putative stem cells on the basis of their functional characteristics the authors stained human
mammosphere cultures with a fluorescent marker, PKH26, which labels quiescent cells but not proliferating
cells as it is lost by dilution. They recovered PKH26-positive (PKH26POS) cells by fluorescence-activated cell
sorting. Unlike PKH26-negative (PKH26NEG) cells, PKH26POS cells formed both basal and luminal cells in two-
dimensional differentiation assays, were able to re-establish mammary development in the cleared fat pads of
immunocompromized mice and possessed other characteristics expected of hNMSCs.
By comparing microarray expression profiles of PKH26POS cells with PKH26NEG cells, a gene signature for
hNMSCs was identified and some of the newly identified stem cell genes could be used as markers to
accurately isolate hNMSCs from normal human mammary glands. Meta-analyses of published breast cancer
gene data sets revealed a correlation between the expression of several candidate signature genes and the state
of tumour differentiation. Poorly differentiated tumours expressed higher levels of genes in the hNMSC gene
signature compared with well-differentiated tumours.
Does the heterogeneity of breast cancers reflect their CSC-like content? Di Fiore and colleagues indeed showed
that CSC-like cells isolated from poorly differentiated tumours using markers from the hNMSC signature set
formed mammospheres in culture and tumour xenografts more efficiently than normal hNMSCs or CSC-like
cells isolated from well-differentiated tumours.
Recently, this group also showed that CSCs more often divide symmetrically to produce daughter stem cells
rather than asymmetrically to produce one stem and one progenitor cell. Together with the findings in this study,
the authors present a model for mammary tumorigenesis in which oncogenic mutations in stem cell populations
determine the frequency at which CSCs will skip asymmetric divisions. This will influence the number of stem
cells in the tumour with resulting changes in the pathological features of the tumour.
References
Pece, S. et al. Biological and molecular heterogeneity of breast cancers correlates with their cancer stem
cell content. Cell 140, 6273 (2010)