Download The Omics of Triple-Negative Breast Cancers

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Clinical Chemistry 60:1
122–133 (2014)
The Omics of Triple-Negative Breast Cancers
Hong Xu,1 Peter Eirew,1,2 Sarah C. Mullaly,1 and Samuel Aparicio1,2
BACKGROUND: Triple-negative breast cancers (TNBC)
do not represent a single disease subgroup and are often aggressive breast cancers with poor prognoses.
Unlike estrogen/progesterone receptor and HER2 (human epidermal growth factor receptor 2) breast cancers, which are responsive to targeted treatments, there
is no effective targeted therapy for TNBC, although approximately 50% of patients respond to conventional
chemotherapies, including taxanes, anthracyclines, cyclophosphamide, and platinum salts.
CONTENT: Genomic studies have helped clarify some of
the possible disease groupings that make up TNBC.
We discuss the findings, including copy number–
transcriptome analysis, whole genome sequencing,
and exome sequencing, in terms of the biological
properties and phenotypes that make up the constellation of TNBC. The relationships between subgroups defined by transcriptome and genome analysis are discussed.
SUMMARY: TNBC is not a uniform molecular or disease
entity but a constellation of variably well-defined biological properties whose relationship to each other is
not understood. There is good support for the existence of a basal expression subtype, p53 mutated, high–
genomic instability subtype of TNBC. This should be
considered a distinct TNBC subtype. Other subtypes
with variable degrees of supporting evidence exist
within the nonbasal/p53wt (wild-type p53) TNBC, including a group of TNBC with PI3K (phosphoinositide
3-kinase) pathway activation that have better overall
prognosis than the basal TNBC. Consistent molecular
phenotyping of TNBC by whole genome sequencing,
transcriptomics, and functional studies with patientderived tumor xenograft models will be essential components in clinical and biological studies as means of
resolving this heterogeneity.
© 2013 American Association for Clinical Chemistry
Department of Molecular Oncology, BC Cancer Agency, Vancouver, BC; 2 Department of Pathology and Laboratory Medicine, University of British Columbia,
Vancouver, BC.
* Address correspondence to this author at: Department of Molecular Oncology,
BC Cancer Research Centre, 675 West 10th Ave., Vancouver, BC, Canada, V5Z
1L3. Fax 604-675-8218; e-mail [email protected].
Received September 25, 2013; accepted November 4, 2013.
Previously published online at DOI: 10.1373/clinchem.2013.207167
Triple-Negative Breast Cancers in the
Clinical Domain
Triple-negative breast cancers (TNBC)3 are clinically
classified by determination of absent estrogen receptor
(ER),3 progesterone receptor (PR), and ERBB2 [human epidermal growth factor receptor 2 (HER2)] receptor status in malignant cells, which is undertaken
during the molecular assessment of a patient’s tumor.
Large population-based surveys of breast cancer prevalence show that TNBCs comprise 16% of breast cancer cases (1 ). TNBC, especially the basal expression
subtype, is more prevalent in young women (⬍50
years) and in women of African or Hispanic descent
(2, 3 ). Because of the lack of specific drug targets,
TNBC is currently treated with conventional, moderately successful chemotherapies, including taxanes, anthracyclines, cyclophosphamide, and platinum salts.
Clinical/pathological features of TNBC include larger
tumor size, higher histological tumor grade, extensive
lymphocytic infiltration, high mitotic counts, and
higher nuclear pleomorphism (4 ). Most TNBCs are of
histological grade III; however, 10% are grade I (2 ),
indicating tumor heterogeneity. TNBC is more aggressive than other breast cancers and displays a nonproportional hazard structure with respect to survival—
TNBC patients have a higher rate of early relapse and
reduced survival, with the peak recurrence happening
between the first and third years, and the majority of
deaths occurring in the first 5 years, following therapy
(5 ). After 5 years, the survival rate of TNBC becomes
similar to that for other types of breast cancer.
Although most TNBCs are classified by histomorphology as ductal NOS (not otherwise specified), it is apparent that some special subtypes such as medullary and
metaplastic breast cancers probably overlap substantially
with the TNBC subtype (6 –10 ). With the exception of
staging and grading, histomorphology is not otherwise
helpful in terms of disease management. Molecular classification is subject to some analytical variation. Whereas
1
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3
Nonstandard abbreviations: TNBC, triple-negative breast cancers; ER, estrogen
receptor; PR, progesterone receptor; HER2, human epidermal growth factor
receptor 2; IHC, immunohistochemistry; PDX, patient-derived xenografts; EGFR,
epidermal growth factor receptor; BL, basal like; EMT, epithelial–mesenchymal
transition; AR, androgen receptor; RTK, receptor tyrosine kinase; LOH, loss of
heterozygosity; HR, homologous recombination; PARP, poly ADP ribose polymerase; VEGF, vascular endothelial growth factor; VEGF receptor 1 (VEGF-1).
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Breast Cancer Genomics
ER and PR are determined by immunohistochemistry,
ERBB2 is determined by a combination of immunohistochemistry (IHC) and/or fluorescence in situ hybridization in North America, according to the American Society
of Clinical Oncology/College of American Pathologists
Her2 testing guidelines (11–13 ). The false positives and
false negatives and variations in IHC that occur in clinical testing regimes for ER and HER2, as well as intratumoral clonal variation, contribute to clinical misclassification in 5%–10% of cases. This variability must be
borne in mind when clinical classification is used in
research studies, without revalidation of the molecular
status. Effective external quality assurance for both the
pathologists and laboratories reading IHC is essential
to reduce misclassification.
MOLECULAR SUBTYPES OF TNBC AND THEIR CAVEATS
There are no accepted international standards for defining new molecular subtypes of cancers, although
some guidelines (14 ) exist for the application of molecular subtyping to prognostic or predictive guidance
in the clinic. A full discussion of these issues is beyond the
scope of this article, but it is especially important for
TNBC, for which many potential markers of molecular
subtypes or biological characteristics have been reported.
The strictest definition would be a “mechanism of
action”-related molecular marker that defines a diseasemodifying clinical intervention, with level 1 evidence (a
prospective, randomized clinical trial). Unfortunately,
there are no such markers for subgrouping TNBCs.
With the caveats noted below, transcriptome analysis in breast cancers has suggested 5 or 6 molecular
subtypes, including the major subtype of TNBC known
as basal expression TNBC (15, 16 ). Recent copy number–transcriptome analysis of breast cancers (all types)
has suggested additional subdivisions (17, 18 ) into at
least 10 IntClust groups (discovered from analyzing
1000 breast cancers and validated by retesting on a second independent cohort of 1000 breast cancers), emphasizing that nonbasal TNBC have a different somatic
mutation spectrum and better overall prognosis than
basal TNBC. In the 10 IntClusters classified by the
combination of copy number variation and gene expression profile, TNBCs are mainly distributed in
IntCluster10 and IntClust4. IntClust4 contains 26%
TNBC and is characterized by low levels of genomic
instability and a copy number aberration– devoid landscape. Somatic T-cell receptor rearrangement coming
from infiltrated mature T lymphocytes is observed in
about 20% of all IntClust4 group tumors (17, 18 ). The
IntClust4 group also has distinct associated microRNA
signatures (19 ) and is associated with good prognosis.
On the other hand, TNBCs in IntCluster10 display high
levels of genomic instability and poor survival in the
first 5 years after diagnosis. This group is enriched for
basal-like TNBC, displaying stereotyped copy number
changes with 5q loss and gains at 8q, 10p, and 12p.
Analysis of mutation frequencies in these 2 groups also
shows subtype-specific patterns. IntCluster10 has the
highest rate of tumor protein p53 (TP53)4 mutations;
in IntCluster4, the most frequently mutated gene is
phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA). Mutations in Usher syndrome 2A (autosomal recessive, mild) (USH2A), filaggrin (FLG), and FAT atypical cadherin 3 (FAT3) are
associated with IntCluster10, and v-akt murine thymoma viral oncogene homolog 1 (AKT1) mutation is
specifically associated with IntCluster4. The heterogeneity within TNBC supports the necessity of applying
different drugs targeting specific driver genes or pathways perturbed within the patient.
Taken together, there is evidence for a major subgrouping of a genomically unstable, basal expression pattern, p53-mutated subgroup of TNBC (Fig. 1); the remaining nonbasal, PIK3CA-enriched TNBCs probably
form several distinct groupings and are of generally better
prognosis. However, all proposed subgroups with intermediate levels of evidence have to be interpreted with
some caution. Currently, clinical outcome associations
with individual molecular markers are very prone to overfitting of data, especially in small groups of patients. Also,
many genomic/transcriptomic markers can be carried together, so the observation of an outcome association is
not in itself proof of a biological subtype with a specific
marker. Functional studies in cell lines are also problematic, because it is clear that the available TNBC cell lines do
4
Genes: TP53, tumor protein p53; PIK3CA, phosphatidylinositol-4,5bisphosphate 3-kinase, catalytic subunit alpha; USH2A, Usher syndrome 2A
(autosomal recessive, mild); FLG, filaggrin; FAT3, FAT atypical cadherin 3; AKT1,
v-akt murine thymoma viral oncogene homolog 1; BRCA1, breast cancer 1, early
onset; MKI67, marker of proliferation Ki-67; TOP2A, topoisomerase (DNA) II
alpha 170kDa; PTEN, phosphatase and tensin homolog; BRCA2, breast cancer
2, early onset; MYO3A, myosin IIIA; RB1, retinoblastoma 1 (Homo sapiens);
ATR, ataxia telangiectasia and Rad3 related; UBR1, ubiquitin protein ligase E3
component n-recognin 1; COL6A3, collagen, type VI, alpha 3; BRAF, v-raf
murine sarcoma viral oncogene homolog B; NRAS, neuroblastoma RAS viral
(v-ras) oncogene homolog; ERBB2, v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 2; ERBB3, v-erb-b2 avian erythroblastic leukemia viral
oncogene homolog 3; MYC, v-myc avian myelocytomatosis viral oncogene
homolog; RAS, rat sarcoma viral oncogene homolog; Rb1, retinoblastoma 1
(Mus musculus); AURKB, aurora kinase B; BCL2, B-cell CLL/lymphoma 2; BUB1,
BUB1 mitotic checkpoint serine/threonine kinase; CDCA3, cell division cycle
associated 3; CDCA4, cell division cycle associated 4; CDC20, cell division cycle
20; CDC45, cell division cycle 45; CHEK1, checkpoint kinase 1; FOXM1, forkhead
box M1; HDAC2, histone deacetylase 2; IGF1R, insulin-like growth factor 1
receptor; KIF2C, kinesin family member 2C; KIFC1, kinesin family member C1;
MTHFD1L, methylenetetrahydrofolate dehydrogenase (NADP⫹ dependent)
1-like; TTK, TTK protein kinase; UBE2C, ubiquitin-conjugating enzyme E2C;
PARK2, parkin RBR E3 ubiquitin protein ligase; EGFR, epidermal growth factor
receptor; FGFR2, fibroblast growth factor receptor 2; PTEN, phosphatase and
tensin homolog; ATM, ataxia telangiectasia mutated; CHEK2, checkpoint kinase
2; UBR5, ubiquitin protein ligase E3 component n-recognin 5; HIFIA, hypoxia
inducible factor 1, alpha subunit (basic helix-loop-helix transcription factor);
ARNT, aryl hydrocarbon receptor nuclear translocator.
Clinical Chemistry 60:1 (2014) 123
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Fig. 1. TNBCs at a glance.
RB1, retinoblastoma 1.
not represent the spectrum of mutations and other
disease-associated events seen in TNBC. Many of these
cell lines are derived from patients with multiply pretreated metastatic disease and also carry the adaptations
necessary for survival in vitro. This makes the translation
of function in a model system to function in situ in patients very uncertain. Studies of patient-derived xenografts (PDX) at different stages of disease can overcome
some of these limitations, but also suffer from clonal adaptation during engraftment. Representative TNBC PDX
are still in their infancy and much larger numbers of characterized xenografts are required. A full discussion of this
is beyond the scope of this review. With all of these caveats
in mind, probably the single most important current observation concerning TNBC is that this group of patients
is quite clearly not a uniform clinical or molecular entity.
124 Clinical Chemistry 60:1 (2014)
Progress will be difficult to come by until clinical studies
and disease mechanism studies also subtype patient tumors and collect the materials on which to rigorously review and reevaluate “subtype,” as new information accrues. Uniform and consistent assays that measure
mutations, expression, copy number, and methylation on
FFPE materials would greatly advance the field by allowing cost-effective and robust genotyping of patients in
clinical studies.
The Cell Types of Origin for TNBC
The phenotype of cancers can be understood as the
product of transforming events (genetic and epigenetic), which take place on the background of a cell of
origin. However, considerable controversy and uncer-
Breast Cancer Genomics
tainty exists as to the cell types of origin of TNBC.
Mammary epithelial lineages (luminal, myoepithelial)
are generated throughout life by a tissue hierarchy that
comprises long-lived, self-renewing, bilineage stem
cells, as well as bilineage, luminal-restricted, and
myoepithelial-restricted progenitor cells that occupy
an intermediate position in the hierarchy (20 –26 ). The
cells with bilineage potential (stem cells and bilineage
progenitors), as well as myoepithelial-restricted progenitors, share many phenotypic markers with basally
located myoepithelial cells (e.g., ␣6-integrinhigh␤1integrinhighEpCAM-/low), whereas progenitor cells
committed to the luminal lineage (both ER⫹ and ER⫺)
express markers that overlap with differentiated luminal cells (e.g., EpCAMhighCD24⫹) (20 –22, 25, 27 ).
The persistence in breast and other cancers of dysregulated developmental hierarchies is supported by studies
showing differential repopulating capacity of sorted
cell fractions when implanted into immunodeficient
mice (28 –32 ).
Key unresolved questions are: how the developmental potential of mammary cells in which transforming mutations arise modulates the impact of those
mutations, and conversely, how specific mutations
perturb the regulation of the hierarchical process itself.
Although such information is mostly lacking in the
majority of human breast cancer subtypes, some progress has been made with studies of BRCA1-deficient
tumors.
Lim and colleagues demonstrated that histologically normal preneoplastic breast tissue in human
breast cancer 1, early onset (BRCA1)-mutation carriers
(i.e., germline-heterozygous) contains increased proportions of ␣6-integrinhighEpCAM⫹ cells (a phenotype
that enriches for luminal progenitors) and fewer cells
in the ␣6-integrinhighEpCAM- fraction (mostly mature
myoepithelial cells, but also stem cells, bipotent cells,
and myoepithelial progenitors) (27 ). Furthermore, the
BRCA1-mutated luminal progenitors are functionally
perturbed, with enhanced growth factor–independent
clonogenic properties in vitro. The gene expression signature of the luminal progenitor-enriched normal cell
fraction correlated more with human basal-type breast
cancers than with the other molecular subtypes (including luminal A and B subtypes, which showed the
lowest correlation). Although suggesting retention of
aspects of the luminal differentiation program within
basal cancers, it is likely that the correlations are partly
influenced by the proliferative nature of the normal luminal-progenitor-enriched fraction, a property shared by highly proliferative breast cancer subtypes, such as BRCA-deficient/other TNBCs.
The interaction of developmental potential with
oncogenic mutations, including BRCA1 loss, has also
been investigated by targeting mutations experimen-
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tally to specific cell subsets. Luminal EpCAM⫹ cells
from normal or BRCA1-mutation– carrying human
mammary tissue were more readily transformed than
basal CD10⫹ cells by a lentiviral oncogene cocktail of
mutated p53, cyclinD1, activated PI3K, and oncogenic
K-ras (33 ). Conditional BRCA1 deletion in p53⫹/⫺
mice resulted in the development of tumors using either Blg-Cre or K14-Cre drivers (the former targeting
mainly ER luminal cells, including luminal progenitors, the latter targeting basal cells, including stem
cells). The histopathology of mouse tumors generated
under the luminal-targeting Blg-Cre more closely resembled human BRCA1⫺/⫺ tumors. Differences were
less evident at the overall transcriptome level, with
both sets of tumors correlating with the human basal
subtype (34 ). The Blg-Cre BCRA1f/f tumors also
closely resembled tumors previously reported in a K14Cre BRCA1f/fp53f/f model (35 ), highlighting the sensitivity of such studies to factors such as genetic background and transgene strategy.
Deconstructing the functional interactions between complex mutational patterns and cellular developmental properties in an evolving tissue hierarchy
remains challenging. The BRCA studies may be interpreted as supporting either luminal progenitor or stem
cell hypotheses of origin, and analogous studies have
not yet been carried out on non-BRCA-deficient tumors (the majority of TNBCs). Key to making progress
may be the development of serial mutation approaches
to model the genetic profile of different TNBC subtypes on different cell types of origin backgrounds, as
well as research to deepen our understanding of the
molecular basis of the properties that define stem and
progenitor function.
Omics-Based Molecular Subgroups of TNBC
Attempts to understand the disease groupings that
make up TNBC have received some impetus in the last
10 years from genome-scale analysis of the transcriptome, genome, and epigenome. Within the last year,
genome sequencing has added to our understanding.
Also, functional screens in cell lines imply some molecular features not evident from the large scale omics.
This is an evolving area in which many of the disease
relationships remain tenuous; we review each, with a
focus on recent genome studies.
TRANSCRIPTOMICS AND THE “BASAL” TNBC SUBTYPE
This aspect of TNBC has been extensively reviewed
elsewhere (36 ). Early microarray studies defined a molecular subtype known as basal (from similarity to the
transcriptome observed in basal myoepithelial cells of
the breast) (15, 16 ), which has a strong overlap with
TNBC status. These early studies have received support
Clinical Chemistry 60:1 (2014) 125
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from multiple independent studies (3, 9, 37– 41 ) confirming basal-like expression characteristics as a reproducible grouping of patients, with discrete patient
survival characteristics. When gene expression data
are compiled from different sources, basal-like tumors in TNBC consistently cluster within a group
that express CK5, 6, 14, 17, and caveolin1/2, as well
as c-kit and P-cadherin (15, 42 ). Approximately
50% of transcriptome-defined basal-like tumors express EGFR. Genes associated with cell proliferation,
such as marker of proliferation Ki-67 (MKI67) (also
known as Ki67) and topoisomerase (DNA) II alpha
170kDa (TOP2A), are also highly expressed in basallike tumors. In parallel with the “core” basal expression
grouping, evidence from the distribution of somatic
p53 and PI3K mutations, copy number analysis, as well
as genome sequencing of TNBC (discussed below),
shows that the basal expression cancers are the most
prevalent/molecularly homogenous subgroup, with a
distinct patient survival characteristic. Thus, basal expression subtype, p53-mutated breast cancers, representing approximately 60%–70% of TNBC, have the
strongest evidence in favor of being a TNBC subgroup.
of differentiation state, and this grouping is not well
defined.
TRANSCRIPTOMICS OF NONBASAL TNBC SUBTYPES
ANDROGEN RECEPTOR PATHWAY IN TNBC
Transcriptomic studies of TNBC have suggested that
other expression-based groupings may exist. The identification of tumors with expression patterns similar to
mesenchymal cells has suggested the possible existence
of such a subgroup, originally named “claudin-low”
(low expression of claudin genes) (43 ), or EMT
signature-enriched TNBC (44, 45 ). The mesenchymal
expression signature has received support from independent studies. A recent metaanalysis of breast cancer
transcriptome studies combined data from 21 publicly
available datasets and 587 TNBC cases of pretreatment
and posttreatment tumors (discovery set 386 cases, validation set 201 cases), and suggested 6 consistent
TNBC subgroups: 2 basal-like (BL1, BL2), an immune
signature group, a mesenchymal group, a mesenchymal stem-like group, and a luminal androgen receptor subtype (46 ). The exact molecular definition of
“TNBC with a mesenchymal pattern” remains uncertain; different gene sets have been proposed in different
studies. It is unclear whether these tumors have distinct
biology in patients, although the expression patterns
have been interpreted as support for epithelial–
mesenchymal transition (EMT) differentiation in
TNBC. For example, recently it has been shown in
mouse-knockout models (47 ) that the combination of
p53 deletion and c-met activation results in mesenchymal expression pattern mouse tumors. Taken together,
there is evidence for an EMT/mesenchymal expression
pattern among TNBC; it is less clear if this is a group of
cancers with distinct biology in patients, or a reflection
Androgen receptor (AR) expression in breast cancers
was first noticed in the mid-1980s as hormone receptor
assays were developed (56 ) and the existence of a subset of ER⫺/PR⫺ breast cancers expressing AR was
noted in 2006 (57 ), with the identification of a AR⫹
breast cell line MDA-MB453, which has androgen responsiveness. The existence of an AR-expressing cancer subset was also noted in a recent metaanalysis of
microarray studies (46 ); nonrecurrent somatic mutations in some AR-responsive genes (although not AR
itself) were noted in the recent sequencing of TNBC
(58 ). These observations have led to interest in the possible efficacy of AR modulators in TNBC. A recently
reported phase 2 trial of bicalutamide (59 ) concluded
that 12% of ER⫺/PR⫺ breast cancers express AR, and a
clinical benefit of 19% was observed for patients in the
treatment arm of this study. This suggests that there
may exist a group of AR pathway-activated TNBC, with
obvious potential for therapeutic intervention. Additional trials and studies are required to determine the
nature of AR activation/responsiveness and to define
the relationships, if any, with other molecular features
of TNBC.
126 Clinical Chemistry 60:1 (2014)
IMMUNE GENE EXPRESSION IN TNBC
Several studies have reported distinct immune gene expression patterns in TNBC (43, 48 –53 ). Some of these
reflect tumors with inflammatory infiltrates, although
it is not clear whether these form a distinct subtype.
Immune signatures have also been associated with
DNA damage as an intrinsic response (54, 55 ) and
some TNBC may have patterns of genome damage resulting in this type of immune signature. The reports of
outcome associations for immune-associated signatures and infiltrates have been contradictory. It is not
clear that TNBC with inflammatory gene signatures
could be considered a discrete subgroup, but the presence of these signatures in some tumors and not others
is a recognized phenomenon which is worthy of further
study to determine whether some TNBC have an associated immune response or inflammatory response. A
possibility that remains to be explored is that TNBC
with intrinsic immune gene expression also have specific patterns of genome damage.
SOMATIC GENOMIC LANDSCAPE OF TNBC
The results from next-generation sequencing of breast
cancers, including whole genome, or whole exome
DNA sequencing, indicate that mutations in breast
cancers show strong subtype-specific segregation
(42, 51, 58, 60, 61 ). In the case of TNBC, the main dis-
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Breast Cancer Genomics
tinction at present is between basal expression subtype
and nonbasal TNBC with respect to the abundance and
types of mutations.
form of INPP4B (68 ), which lacks a phosphatase domain in cells with a basal-like expression program.
BRCA1 AND BRCA2
P53 pathway. Mutations in TP53 are highly prevalent in
basal-like TNBC. The reported prevalence varies,
probably due to differences in classifying tumors and in
part due to ascertainment, with 60%– 80% of basal
TNBC reported (18, 42, 58, 62– 64 ) as having p53 mutations. Recently it has been suggested that basal tumors are enriched for p53 nonsense and frameshift
mutations (as opposed to point mutations, which are
activating). A frequent occurrence is loss of heterozygosity (LOH) affecting one allele of p53, which may
combine with methylation/mutation leading to inactivation of the second allele. Analysis of allele-specific
expression between the tumor RNA and DNA has
identified TP53 with strong transcriptional allelic imbalance, preferentially expressing only mutant TP53 alleles (65 ).
PI3K pathway. Mutations in the PI3K pathway represent the next most prevalent somatic mutation
(42, 51, 58, 60 ), and these tend to occur more frequently in the nonbasal TNBC, although this is not a
strict relationship. Mutations in PIK3CA are approximately 10% for TNBC, although the overall mutation
frequency in all breast tumors for PIK3CA (36%) is
comparable with TP53 (37%). Copy number analysis
also revealed significant focal amplifications in PIK3CA.
PIK3CA mutations and copy number gains lead to the
activation of the PI3K pathway. The recurrent PIK3CA
E545K mutation presents most exclusively in the luminal A subtype, rather than in basal TNBC. An alternative mechanism leading to activation of the PI3K pathway is the loss of phosphatase and tensin homolog
(PTEN) through mutation, with a prevalence in TNBC
of approximately 7.7%. Additional PTEN silencing
may occur epigenetically, and thus combined, the PI3K
pathway may be activated in approximately 15%–20%
of TNBC, with enrichment in the nonbasal expressiontype tumors. However, trials of PIK3CA agents have
not yet shown a major impact, perhaps in part because
of inadequate initial patient selection. PIK3CA/PTENmutated TNBC tumors tend to group with better prognosis breast cancers in the revised IntClust groupings
(18 ), rather than with the basal expression subtype. Another aspect of PI3K-Akt activity that may predominate in basal-expression type TNBC is the regulation of
phosphatase expression. The phosphatase INPP4B has
recently been implicated as an important regulator of
PIP3 signaling, and expression of full-length INPP4B is
very low in a majority of basal-expression subtype
TNBC (66, 67 ). This may be the result of LOH at the
locus, or the constitutive expression of the short iso-
BRCA1 or breast cancer 2, early onset (BRCA2) germline mutation carriers have a high probability (70%–
80%) of developing breast cancer (69 ) and a higher risk
for ovarian cancer (30%–50%). The relative risk for
developing other types of cancer is also greatly increased. Whereas breast cancers from BRCA2 carriers
are frequently ER⫹, tumors from BRCA1 carriers tend
to be TNBC. However, if care is taken to eliminate previously undiagnosed BRCA1 germline carriers, somatic mutations in BRCA1 are infrequent in sporadic
TNBC patients (42, 58 ).
TNBC patients may inactivate the BRCA1/2 pathways by methylation or downregulating BRCA function. For example, the protein ID4 is overexpressed and
significantly unmethylated in TNBC, possibly accounting for repression of BRCA1 at the protein level (70 ).
Once germline carriers are accounted for, nonmutational loss of expression may occur in perhaps 10%–
15% of TNBC (42, 58 ). Mutational patterns suggest
that recombination defects may occur even in non–
BRCA mutated breast cancers (71 ), indicating the possible relevance of the homologous recombination
(HR) pathway (72 ). Convincing evidence of functional
loss of HR-repair capacity in a significant number of
sporadic TNBC patients is lacking, and the support
from therapeutic responses to drugs targeting these
pathways in TNBC is mixed. A recent phase 2 trial of a
poly ADP ribose polymerase (PARP) inhibitor in sporadic ovarian and TNBC (73 ), showed no activity in
the TNBC arm, suggesting “BRCAness” may not be a
frequent phenotype in TNBC. In contrast, clinical reports of responses to platinum salts (74 ) may suggest
that some TNBC patients have a functional deficiency
in HR, which could be exploited. It is possible that the
nonmutational mechanisms result in hypomorphic
BRCA function, sufficient to moderately impair HR,
but not sufficient to confer sensitivity to agents targeting this deficiency. It is hard to estimate how many
TNBC patients fall into this group, but it is probably
not more than 20% of all TNBC. Larger unbiased studies of patients with TNBC that clearly distinguish between germline and somatic mutations and that account for all other possible mechanisms of inactivation
recombination repair are required.
ADDITIONAL SOMATIC MUTATIONS IN TNBC
Other significantly mutated single genes reported in
TNBC include: USH2A, myosin IIIA (MYO3A), PTEN,
retinoblastoma 1 (RB1), ataxia telangiectasia and Rad3
related (ATR), ubiquitin protein ligase E3 component
n-recognin 1 (UBR1), collagen, type VI, alpha 3
Clinical Chemistry 60:1 (2014) 127
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(COL6A3), and several well-known oncogenes [v-raf
murine sarcoma viral oncogene homolog B (BRAF),
neuroblastoma RAS viral (v-ras) oncogene homolog
(NRAS), v-erb-b2 avian erythroblastic leukemia viral
oncogene homolog 2 (ERBB2), and v-erb-b2 avian
erythroblastic leukemia viral oncogene homolog 3
(ERBB3)] (18, 42, 58 ). Activating mutations of
ERBB2, rare in TNBC, may present an opportunity for
intervention with targeted kinase inhibitors (75 ). The
majority of the significantly mutated genes in nonTNBC subtypes, except TP53 and PIK3CA, are only
rarely observed in basal-like TNBC, indicating a
subtype-specific mutational spectrum.
The distribution of allele prevalence in TNBC has
a long tail; most somatic mutations are observed once,
and current statistical methods are not well suited to
assessing the relevance of these mutations. Prevalencebased mutation assessment in a patient population is
weighted toward the discovery of early founder mutations that dominate the clonal structure of a tumor.
Approaches for deciphering somatic mutations implicated in progression are less well established. One approach is to group mutations by virtue of the known
functions of the genes in which they occur, using systematic network analysis. Another approach is to
search for mutated genes with a disproportional effect
on the transcriptional network known to associate with
the gene in question (76 ). Using these approaches and
combining copy number effects and nonmutational alterations in expression, additional genes have been implicated in TNBC, including the RB1, v-myc avian myelocytomatosis viral oncogene homolog (MYC), and
rat sarcoma viral oncogene homolog (RAS) gene networks. RB1 may have special significance in potential
contributions to the cell cycle abnormalities and
genomic instability in TNBC (77–79 ). The cooccurrence of dysfunction in these networks with respect to
the basal/nonbasal phenotype is less clear. In mice, retinoblastoma 1 (Rb1) gene loss of function has been
associated with both luminal and basal tumors
(77, 78 ). Overexpression of MYC has been noted in
breast cancers, and frequent amplification of the c-myc
locus on chromosome 8 has been observed, although
this does not always result in increased expression (18 ).
Some studies have suggested that Ras activation is
functionally important in breast cancer, however RAS
is not frequently affected [⬍5% of all breast cancers
(42, 51, 58, 60, 61 )] by oncogenic mutational mechanisms in breast cancers. Better-controlled studies are
needed to define the subtype-associated relevance of
these pathways.
Recent genome sequencing of primary TNBC (58 )
revealed a distributed pattern of somatic mutations in
genes implicated in cell shape and motility, and the
extracellular matrix. No single gene was mutated at
128 Clinical Chemistry 60:1 (2014)
⬎5% prevalence, with the exception of MYO3A; however, the gene family and biological process was significantly overrepresented. The extracellular matrix plays
a key role in the maintenance of the tumor microenvironment and contributes to tumor progression/metastasis through the promotion of angiogenesis, tumorassociated inflammation, and EMT. Actins are also
involved in this process and implicated in invasion/
survival processes. Whether this pattern represents
functional mutations in this family of proteins, or some
other process, such as transcription-dependent repair/
mutation, is unknown.
COPY NUMBER AND CHROMOSOMAL LEVEL GENOME
ABERRATIONS
TNBC are characterized by extensive genomic instability. Basal expression-type TNBC have significantly more genomic instability than nonbasal
TNBC (18, 58, 80, 81 ). This may be a reflection of
early p53 inactivation, or aberrant expression of
c-myc by nonmutational mechanisms; however, mutation or aberrant expression of genes implicated in cell
cycle checkpoints (RB1), cell division mechanisms (cyclin amplification), and genome maintenance (homologous recombination, PTEN loss), could all contribute
to the landscape.
High genomic instability in basal-like tumors at
the chromosome structural level has been reported
(82, 83 ). Recurrent large-scale chromosomal changes
have been noted for basal cancers, with loss of 5q and
gain on 10p being somewhat characteristic of basal
cancers. 5q loss may be associated with basal-specific
transcription patterns, as demonstrated by in trans
analysis of copy number and expression (18 ). This
method searches for expression relationships to copy
number at other than the canonical gene or region (defined as in cis). In the recent reclassification of breast
cancers on the basis of copy number expression analysis, the 5q loss is associated with a strong basal-specific
signature, including aurora kinase B (AURKB), B-cell
CLL/lymphoma 2 (BCL2), BUB1 mitotic checkpoint
serine/threonine kinase (BUB1), cell division cycle associated 3 (CDCA3), cell division cycle associated 4
(CDCA4), cell division cycle 20 (CDC20), cell division
cycle 45 (CDC45), checkpoint kinase 1 (CHEK1),
forkhead box M1 (FOXM1), histone deacetylase 2
(HDAC2), insulin-like growth factor 1 receptor
(IGF1R), kinesin family member 2C (KIF2C), kinesin family member C1 (KIFC1), methylenetetrahydrofolate dehydrogenase (NADP⫹ dependent)
1-like (MTHFD1L), TTK protein kinase (TTK), and
ubiquitin-conjugating enzyme E2C (UBE2C). The precise 5q driver gene(s) remain unknown, but this recurrent chromosomal loss requires further investigation
to define the basal-specific signature (17 ). Monoallelic
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Breast Cancer Genomics
expression associated with LOH is frequent in TNBC
and has been mapped to high resolution (84 ).
Recurrent focal amplifications and deletions affecting gene expression are uncommon in TNBC, but a
few have been observed. Parkin RBR E3 ubiquitin protein ligase (PARK2) was reported as a tumor suppressor in colorectal cancer and is a newly discovered tumor suppressor in TNBC. Intragenic deletion in this
gene is found in 6% of the TNBC tumors. Other genes
affected by copy number alteration events are RB1
(5%), epidermal growth factor receptor (EGFR) (5%),
fibroblast growth factor receptor 2 (FGFR2) (3%), and
phosphatase and tensin homolog (PTEN) (3%)
(7, 18, 58, 81, 84, 85 ). For patients with these uncommon events, the tumor phenotype may be skewed by
the amplicons/deletion and some (e.g., EGFR, FGFR2)
may represent “actionable” events. Different mechanisms likely shape the mutation landscape and copy
number variation as these are uncorrelated in extent.
Individual breast cancers of all subtypes average
approximately 5 nonrecurrent fusion genes per tumor
sample (86 ), and this is even more prevalent in TNBC.
However, recurrent translocation/fusions are not a feature of breast cancer, although reports of rare recurrent
fusions with Notch (86 ) and Akt3 (87) have been described. In TNBC, Akt3-MAGI3 fusions have been reported to upregulate the expression of AKT3, while
removing the PDZ [PSD95 (postsynaptic density protein), Dlg1 (Drosophila disc large tumor suppressor),
and zo-1 (zonula occludens-1 protein)] domain of
MAGI3, required for PTEN binding. This translocation leads to a combined effect of overexpression oncogene (AKT3) and loss of tumor suppressor (60 ), predicting activation of the PI3K pathway.
EPIGENETIC REGULATION
On the basis of DNA methylation analysis in 802 breast
tumors, TCGA breast tumors are classified into 5
methylation groups. Group 5-represented (basal-like
tumors) has the lowest level of DNA methylation, and
group 3-represented (luminal B subtype) showed hypermethylated phenotype. A study of 91 TNBCs found
significant hypomethylation of stem cell markers
CD44, CD133, and MSH1, confirmed by IHC (87 ).
The functional relationship of global CpG hypomethylation to the basal TNBC phenotype remains to be
explored.
MicroRNAS
MicroRNA expression correlates with cell differentiation and tumor developmental status. It also reflects
the specific pathological features of breast cancer, such
as ER and PR expression, lymph node status, vascular
invasion, proliferation and tumor stage. miR-21 and
miR-221, which play roles in cell growth and prolifer-
ation, are significantly higher in TNBC vs normal tissue. miR-221 also inhibits the expression of ER at the
protein level. miR-210, which increases the expression
of hypoxia-inducible factors, hence promoting tumor
progression in hypoxia, is overexpressed in TNBC. By
contrast, microRNAs that suppress cell proliferation,
such as miR-145 and miR-205, are underexpressed in
TNBC. miR-205 also inhibits the EMT transition and
suppresses tumor expansion from the basal membrane
to the stroma (88 ). Thus, the expression profile of
microRNA associates with the highly proliferative and
high EMT feature of some TNBC.
CLONAL STRUCTURE WITHIN PRIMARY TNBC
Next-generation sequencing methods have opened up
the estimation of clonal composition in human tumors
(reviewed in (89 ). Recently, the clonal complexity of
primary TNBC was assessed for the first time using
these methods (58 ). Using population prevalence
methods of clonal estimation, the number of distinct
clonal groups in primary TNBC was noted to vary
widely from patient to patient at the time of diagnosis.
The tumors were of a similar stage and grade, and all
pT2 or smaller. These patients were treated with
broadly similar regimens, yet their tumors varied in
complexity from 2–3 discernable clonal populations to
7⫹. Basal expression-type TNBC were more clonally
complex than nonbasal, in keeping with the observation that they have a higher mutational burden. Analysis of clonal prevalence (abundance of each clonal
group) by gene function showed that most “founder”
type mutations, such as p53, were clonally prevalent,
whereas somatic mutations in genes functionally associated with progression phenotypes (such as invasion,
discussed above), were of lower clonal prevalence. In
some cases, it was noted that founder mutations such as
p53 were not the most prevalent clone, suggesting that
earlier mutations may have provoked the genomic instability in some tumors. Future analysis of clonal
abundance of low-prevalence mutations in relation to
relapse/progression will be needed to determine
whether clonal analysis can become predictive of relapse potential in primary cancers.
Functionally Implicated Genes and Pathways
Beyond the genes and pathways directly implicated by
mutational mechanisms or epigenetic regulation, additional pathways of potential relevance to TNBC have
been reported. The relationship of these to TNBC subtypes remains mostly tenuous, as the majority of studies are either small (relative to the diversity of the patient population) or have not been rigorously tested
against the omics-based subgroups. The functional
Clinical Chemistry 60:1 (2014) 129
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properties of TNBC have been reviewed extensively
elsewhere and are only summarized here.
RAF-MEK1/2-ERK1/2 PATHWAY
Activated RAF-MEK1/2-ERK1/2 signaling has been
noted in TNBCs, although mutations are seen in ⬍5%
of cases. Clinical trials with MEK inhibitors have failed
because of drug-induced activation of alternative survival signaling pathways (90 ). MEK inhibitor-induced
receptor tyrosine kinase (RTK) stimulation overcame
MEK2 inhibition, reactivating ERK and resulting in
drug resistance. In cell-based assays, the combination
of the MEK inhibitor AZD6244, and the RTK inhibitors sorafenib or foretinib, are synthetic-lethal.
PTPN12 AND OTHER PHOSPHATASES
Tyrosine phosphatase PTPN12 is a putative tumor
suppressor, and loss of PTPN12 protein expression is
prevalent in TNBC (91 ). PTPN12 inhibits the EGFR/
HER2-mitogen-activated protein kinase signaling
pathway to suppress tumor transformation. PTPN12
depletion leads to increased phosphorylation of EGFR
and HER2, and multiple RTKs in TNBC. Combinatorial inhibition of HER2 and PDGFR-␤ by lapatinib and
sunitinib greatly reduces the growth of TNBC, suggesting that PTPN12-deficient TNBC may be successfully
treated with a combination of TK inhibitors. It also
demonstrates that in TNBC, the HER2 pathway can be
activated at the phosphorylation level by PTPN12 deletion without HER2 amplification. It is noteworthy
that the 5q deletion in TNBC, which drives a large in
trans expression network (see above), encompasses
several phosphatases.
ness of chemotherapy. Synthetic lethality is another
strategy currently under investigation for tumors with
loss of function mutations or hypomorphic alleles in
DNA damage repair.
ANGIOGENESIS, HYPOXIA, AND SERINE ADDICTION
Angiogenesis is a hallmark of tumor development.
TNBC express higher levels of vascular endothelial
growth factor (VEGF), the predominant mediator of
angiogenesis (95 ). Acting as a growth factor ligand,
VEGF binds to the RTKs VEGF receptor 1 (VEGFR-1)
and VEGFR-2. Biological options for targeting angiogenesis include: an anti-VEGF monoclonal antibody,
such as bevacizumab, and RTK inhibitors, such as the
sunitinib and sorafenib. Hypoxia inducible factor 1,
alpha subunit (basic helix-loop-helix transcription factor) (HIF1A)/aryl hydrocarbon receptor nuclear translocator (ARNT) pathways, required for angiogenesis,
are also activated in basal-like tumors, suggesting that
HIF1 inhibitors or bioreductive drugs that become activated under hypoxic conditions may benefit patients
with basal-like cancers.
Growth factor pathway activation and hypoxic tumor cell metabolic responses have been noted in
TNBC, and predominantly in basal expression type
cancers. This is reviewed elsewhere, but probably also
forms an important metabolic consequence of transformation (96 –98 ). Targeting hypoxia with CA9 inhibitors is being explored. Serine biosynthesis may be
another key feature, with addiction to serine and its
metabolites being a reported phenotype of cell lines
with basal-expression phenotypes (99 ).
Conclusions
EGFR PATHWAY
EGFR high-level expression is detected (27%–57%) in
TNBC by IHC (92 ). Mutations in EGFR are associated
with extreme changes in transcription of interacting
genes (58 ). Although TK inhibitors have been successfully used to treat EGFR-mutant lung cancers, the effectiveness of EGFR-targeted agents in combination
with chemotherapy in TNBC has been inconsistent and
trials are ongoing (93, 94 ).
NON-BRCA DNA DAMAGE REPAIR PATHWAY
A number of genes in the DNA damage response pathway [ataxia telangiectasia mutated (ATM), ATR,
checkpoint kinase 2 (CHEK2), and ubiquitin protein
ligase E3 component n-recognin 5 (UBR5)] are implicated at low prevalence by mutational mechanisms, but
possibly more frequently by altered expression in
TNBC. Because of the importance of these genes in
DNA damage repair, the primary rationale for drug
design is targeting these genes in combination with the
presence of genotoxic agents to increase the effective130 Clinical Chemistry 60:1 (2014)
Integration of omics data on TNBC is beginning to
shed some light on the constellation of pathologies associated with these breast cancers. Better models,
which are rigorously mapped at the omic level and that
relate the actual behavior and phenotypes of TNBC in
patients, are needed. It is realistic to believe that more
robust molecular hypotheses will be developed on the
basis of this information and will guide further focused
study to better understand TNBC and target these
cancers.
Author Contributions: All authors confirmed they have contributed to
the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design,
acquisition of data, or analysis and interpretation of data; (b) drafting
or revising the article for intellectual content; and (c) final approval of
the published article.
Authors’ Disclosures or Potential Conflicts of Interest: No authors
declared any potential conflicts of interest.
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Breast Cancer Genomics
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