Journal of Cancer Therapy, 2012, 3, 649-654 Published Online October 2012 ( 649
p53 Expression in Triple Negative Breast Carcin omas:
Evidence of Age-Related and Racial Differences*
Simone Davion, Megan Sullivan, Stephen Rohan, Kalliopi P. Siziopikou#
Department of Pathology, Breast Pathology Section, Northwestern University Feinberg School of Medicine, Chicago, USA.
Received May 31st, 2012; revised June 30th, 2012; accepted July 10th, 2012
Triple negative breast carcinomas (TNBC), are defined by the absence of estrogen receptor (ER), progesterone receptor
(PR) and human epidermal growth factor receptor 2 (HER2) expression. The majority of TNBC are “basal-like”, a
group originally defined by studies of mRNA gene expression profiles, but increasingly defined in the clinic by using
surrogate markers such as CK 5/6. However, not all TNBC are basal-like. It is postulated that these subcategories of
TNBC have distinct underlying biologies that drive their ultimate behavior, and response to treatment, with important
implications for designing appropriate targeted therapies. In this study we report that within our cohort of 197 TNBC,
distinct groups were identified that varied by CK 5/6 and p53 expression based on age and race. We propose that
awareness of CK5/6 and p53 expression in younger and AA TNBC patients may facilitate identifying patients with
unique tumor subtypes and may lead to better use of targeted therapies in this group of aggressive breast cancers.
Keywords: Triple Negative Breast Cancer; p53; Race; Age
1. Introduction
Triple negative breast carcinomas (TNBC) represent 10%
- 17% of breast carcinomas and are defined immuno-
histochemically by lack of expression for estrogen re-
ceptor (ER), progesterone receptor (PR) and human epi-
dermal growth factor receptor 2 (HER2) [1]. TNBC are a
heterogeneous group of tumors with different pathologic
characteristics, molecular alterations, clinical features
and biologic behavior. Basal-like breast tumors were so
named because they express many of the genes found in
normal breast myoepithelial cells including cytokeratin
filaments 5, 14, and 17 . Man y of these “basal-like” breast
cancers also have low expression of ER as well as genes
co-expressed with ER. Basal-like tumors represent be-
tween 50% - 75% of TNBC [2-4]. However, breast can-
cers from each intrinsic molecular subtype, including
luminal A, luminal B, Her-2-enriched and claudin-low,
have also been identified within the triple negative group .
Triple negative tumors are most often high grade infil-
trating ductal carcinoma of no special type, exhibiting
rapid growth, often declaring themselves before mam-
mographic screening has begun in young women or in
between mammograms (interval cancers) in older women
[1]. TNBC are not amenable to targeted therapies with
either hormone antagonists, such as tamoxifen, or anti-
bodies targeting HER2 such as trastuzamab. These tu-
mors often respond initially to classes of more indis-
criminate chemotherapies but have a high likelihood of
recurring or progressing [5]. Recently the use of PARP-1
inhibitors in a subgroup of basal-like TNBC has been
entertained and thus accurate subclassification of TNBC
into basal-like and other subtypes is becoming more
clinically relevant [6].
The p53 protein exercises control over cell cycle pro-
gression via its role as a transcription factor that regu lates
expression of a large number of genes which govern cell
cycle arrest and progression. Over half of human cancers
acquire p53 mutations during malignant transformation
resulting from either loss of function or gain of function
mutations. Gain of function mutations in p53 result from
a change in a single amino acid in the DNA binding do-
main, leading to inactivity, increased stability, and ex-
pression of high levels of p53 protein detectable by immu-
nohistochemistry [7]. Immunohistochemical staining for the
p53 protein, performed on tissue sections, is a proxy for gain
of function mutations in p53 that result in inactiv ity.
*The authors have no financial disclosures, conflicts of interest or ack-
nowledgem ents to report.
This study was conducted after approval by the Institutional Review
Board (IRB) of Northwestern University and complies with the US
olicies on ethical consent.
#Corresponding author.
Mutations in the p53 gene have been found to be more
common in basal-like cancers than other breast cancer
subtypes and may interact with retinoblastoma protein
RB1 in claudin-low TNBC [8-10]. p53 gain of function
Copyright © 2012 SciRes. JCT
p53 Expression in Triple Negative Breast Carcinomas: Evidence of Age-Related and Racial Differences
mutations and consequent protein expression, within the
group of triple negativ e breast can cers, has been found to
meaningfully stratify this heterogeneous group of breast
cancers into subtypes with better or worse progno sis, and
differing response to chemotherapy [11,12].
Demographically the basal subtype of TNBC has been
demonstrated to more commonly affect young African
American (AA) women. Hypotheses have been put for-
ward that these biologic differences may explain the
lower incidence but higher mortality of breast cancer in
this cohort of breast cancer patients [4,13-15].
In this study we evaluated the role of p53, a mediator
of cellular response to DNA damage, in a high risk group
of breast cancers with limited therapeutic options, as it
relates to race and age in a cohort of patients diagnosed
at a single academic medical center between 2003-2010.
2. Materials and Methods
This study was conducted after approval of the institu-
tional review board at Northwestern University. The
electronic pathology laboratory information system was
searched for cases of breast carcinoma diagnosed be-
tween 2003-2010. Inclusion criteria were carcinomas
with documented negativity for ER, PR, and HER2, and
the availability of paraffin blocks with representative
tumor and sufficient tissue for immunohistochemical
studies. To be defined as a triple negative carcinoma, the
tumor had to show <1% nuclear positivity with estrogen
receptor and progesterone receptor and be negative for
HER2 by either immunohistochemistry (score 0 or 1+) or
FISH analysis. Pathologic tumor characteristics included
histological type, tumor grade, tumor size, presence of
lymphatic/vascular invasion (LVI), lymph node status
and p53 expression. At our institution p53 expression by
IHC is performed routinely on all newly diagnosed inva-
sive carcinomas of the breast. Cases were considered to
be positive for p53 when >10% of tumor cells demon-
strated nu clear st a ining.
Electronic medical records were reviewed to deter-
mine patient demographics including age and race. Race
was determined either by self-identification at the time of
initial visit, or designated by the clinician at the initial
intake visit. For purposes of analysis race was catego-
rized as Caucasian, African American, Asian, Hispanic
or Other.
2.1. Tissue Microarray Construction
Ninety-nine cases of TNBC with more than one tumor
block available were used to construct tissue microarrays
(TMAs). The TMAs were constructed from the formalin
fixed paraffin embedded tissue blocks. For each patient,
three separate 0.2 cm tumor cores were included to ac-
count for tumor heterogeneity. Embedded within each
TMA were additional cores of positive and nega tive con-
trol tissue.
2.2. Immunohistochemical Studies
Immunohistochemical studies were carried out on the
TMAs with antibodies to cytokeratin 5/6 (DAKO D5/
16b4). Staining for CK 5/6 was carried out at 1:25 solu-
tion after retrieval with mild cell cond itioning solution 1.
Positive control tissue both embedded within the TMA
and on separate slides was evaluated to ensure that the
results were interpretable. CK 5/6 was determined to be
positive with any d egree of positive cytoplasmic stainin g
in accordance with the literature [16].
3. Statistical Analysis
The data were analyzed with cross tabulations and the
chi-squared test was used as appropriate. p-values < 0.05
were considered statistically sig nificant.
4. Results
4.1. Clinicopathologic Data
One hundred and ninety-seven cases of TNBC were re-
trieved from the electronic laboratory information system.
Patient age at diagnosis ranged from 29 - 93 (mean age:
54). Of the 197 TNBC 45/197 (22.8%) were AA,
142/197 (72.0%) were Caucasian, 4/197 (2%) were His-
panic. 2/197 (1%) were Asian, and 4/197 (2%) patients
did not have racial information recorded in the electronic
medical record.
TNBC were almost exclusively infiltrating ductal car-
cinomas of no special type (181/197, 92.3%). The re-
maining cases were either invasive lobular carcinoma
2/19 (1%), metaplastic carcinoma 13/197 (6.6%), or
adenoid cystic carcinoma 1/197 (p = 0.05%) Using the
modified Bloom Richardson grading system, the majority
of the ductal and lobular carcinomas were grade 3 tumors
(150/183, 82%) , and the remain in g w ere g rad e 2 (33 /183 ,
18%). None of the tumors were histologic grade 1 (Table
Table 1. Histologic and breast marker characteristics of the
patient population.
Tumor characte ristics (n = 197)
ER negative 100%
PR negative 100%
HER-2 negative 100%
Ductal histology 92.3%
High histologic grade (III) 82.7%
p53 expression 54.7%
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p53 Expression in Triple Negative Breast Carcinomas: Evidence of Age-Related and Racial Differences 651
4.2. Correlation between p53 Expression and
Overall 67 patients who had their race recorded in the
electronic medical record (18 AA and 49 Caucasian pa-
tients) had their p53 status available. Amongst TNBC
p53 expression was associated with African-American
(AA) race. Specifically more than three quarters 14/18
(77.7%) of AA patients with TNBC expressed p53 com-
pared to just over half 28/49 (57.1%) of Caucasians (p <
0.01) (Table 2). p53 status was not associated with the
tumor grade, tumor size, presence of LVI nor lymph
node status.
4.3. Correlation between Basal Phenotype and
p53 Expression
The basal phenotype of TNBC as defined by cytokeratin
5/6 expression was then correlated with expression of
p53. 83 cases had both p53 and CK 5/6 data available.
p53 was expressed in a higher percentage of basal phe-
notype (CK 5/6 positive) cases ( 29/48, 60.4%) compared
to just over half of the CK 5/6 negative cases (18/35,
51.4%) (Table 3).
4.4. Correlation between Age and CK
5/6-Positive (Basal)/p53-Positive Phenotype
Of interest, of the 83 cases of TNBC that had both p53
and CK 5/6 status available, twice as many (50%) of the
TNBC patients younger than 50 expressed the basal/p53
positive phenotype compared with only 23.4% of the
TNBC patients older than 50 (p < 0.01), independent of
race (Table 4 and Figure 1).
Table 2. Correlation between race and p53 expression.
p53 Expression
Race Positive Negative Total
African-American 14 (77.7%) 4 (22.3%) 18
Caucasian 28 (57.1%) 21 (42.8%) 49
Total 42 25 67
p < 0.01.
Table 3. Correlation between basal phenotype and p53 ex-
p53 Expression
CK5/6 Expression Positive Negative Total
Positive 29 (60.4%) 19 (39.5%) 48
Negative 18 (51.4%) 17 (48.5%) 35
Total 47 36 83
Table 4. Correlation between age and CK 5/6-positive
(basal)/p53-positive phenotype .
Age Phenotype Present Phenotype Absent Total
<50 yo18 (50%) 18 (50%) 36
>50 yo11 (23.4%) 36 (76.6) 47
Total 29 54 83
p < 0.01.
5. Discussion
Our study of a cohort of TNBC diagnosed at a single
academic medical center between 2003-2010, shows that
p53 expression is seen in over half of TNBC and that
TNBC patients younger than 50 years of age are twice as
likely to have p53 expressing TNBC of the basal-like
phenotype than patients older than 50 years of age, re-
gardless of race. Results from our cohort also showed
that the basal phenotype/p53 positive TNBC is more
common in African Americans compared to Caucasians.
Our results are consistent with the Carolina Breast
Cancer study and others [4,15,17,18]. Awareness of these
associations between intrinsic biologic differences and
different tumor phenotypes, as well as the prevalence of
different tumor phenotypes in different ethnic groups is
becoming more important for successful application of
individualized targeted therapies.
It is well documented that African American women
with breast cancer have a poorer prognosis than Cau-
casians. The relative risk of death for African American
women with breast cancer compared to Caucasian pa-
tients is between 1.10 and 1.22, and while the overall
incidence rates for breast cancer amongst African
American is lower than Caucasian women, 128.95 com-
pared to 114.12 per 100,000, the 5 year survival rate for
African Americans is lower than Caucasians, 91.4% ver-
sus 77.4% [19,20].
Many explanations for this disparity have been prof-
fered including variation in stage at diagnosis, treatment,
and access to care, but even after controlling for such
variables African American women still have a higher
mortality rate from breast cancer than Caucasians [21].
Several groups have reported that the biology of breast
cancers in African Americans is different, with African
American women more commonly having triple negative
breast cancers than their Caucasian counterparts [14,15,
Similarly premenopausal women with breast cancer
have lower 5 year survival rates than post-menopausal
women with breast cancer, 83% versus 90% [22,23].
The biology of breast cancers in younger women appears
to be distinct based on histopathologic, immunohisto-
chemical, and mRNA gene expression analysis. Breast
Copyright © 2012 SciRes. JCT
p53 Expression in Triple Negative Breast Carcinomas: Evidence of Age-Related and Racial Differences
Figure 1. Infiltrating ductal carcinoma grade III/III, ER-
negative/PR-negative/HER2-negative exhibiting strong CK
5/6 positivity and high p53 expression.
cancers in younger women is more often of higher grade,
larger size, with more frequent lymph node positivity.
Gene expression profiles of breast cancer in younger
women demonstrates that these cancers have lower levels
of ERα and progesterone receptor expression, with
higher expression of gene subsets related to stem cells
and BRCA1 [24,25]. Tumors in younger women have
also been shown to be less responsive to treatment [22,
The p53 tumor suppressor gene has long been recog-
nized as an important regulator of the normal cell cycle
and mutations in p53 are a major factor in malignant
transformation and tumor progression. Over half of hu-
man cancers acquire p53 mutations during malignant
transformation resulting from either loss of function or
gain of function mutations. p53 mutations have been
shown to be prognostic in breast cancer. Patients with
p53 mutated breast cancers have worse disease free sur-
vival, independent of other risk factors and p53 muta-
tions have been hypothesized to play a role in resistance
to certain chemotherapies [25-27]. Emerging studies are
attempting to elucidate the correlation between p53 mu-
tations and mutations in retinoblastoma protein (RB1) in
TNBC and the claudin-low tumors. The claudin-low tu-
mors are associated with tumor cells that may possess
stem cell qualities such as the ability for self-renewal and
resistance to chemotherapy [10,28].
At this time, routine use of molecular markers such as
p53 and CK 5/6 in defining subgroups of breast cancer
patients, although occasionally reported, is not estab-
lished pathology practice. However, as the association
between p53 mutations, basal subtype, BRCA1 muta-
tions, and the emerging sensitivity of BRCA1 deficient
tumors to PARP inhibitors is beginning to be established,
and the importance of p53 is once again taking center
stage [2].
Furthermore gene microarray analysis has shown that
breast carcinomas can be further classified by their p53
status and this “p53 signature” predicts mutation status
and patient survival in different patient subsets inde-
pendent of other risk factors [27]. Of interest, combined
mutations in retinoblastoma gene RB1 and p53 in mam-
mary stem cell progenitors is reported to induce epi-
thelial to mesench ymal transi tion (EMT) in ani mal mode l s
[10]. In triple negative mammary tumors in mice the
EMT-type tumo r an d a sub set o f ba sal phenotyp e tu mors ,
are reported to express mutant forms of p53, suggesting
that p53 plays a major role in dictating tumor subtype
after RB inactivation in triple negative breast cancers
[29]. It is tempting to speculate that these pathways are
likely to also play a major role in the pathogenesis of
TNBC in humans and are possibly important in deter-
mining respon se to therapy and overall prognosis.
Finally, as therapies targeting the specific subtypes of
TNBC are introduced into wider clinical use, expansion
of the routine panel of immunohistochemical tumor
markers performed on invasive breast cancers to include
p53 and basal-type markers may be necessary. Aware-
ness of the increased likelihood of finding these tumors
in TNBC patients younger than 50, as well as AA pa-
tients may ultimately lead to successful individualized
regimens for these difficult to treat subtypes of breast
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p53 Expression in Triple Negative Breast Carcinomas: Evidence of Age-Related and Racial Differences
AA: African American; TNBC: Triple Negative Breast
Carcinoma; CK 5/6: Cytokeratin 5/6; LVI: Lymphatic
Vascular Invasion; ER: Estrogen Receptor; PR: Pro-
gester-One Receptor; HER2: Epidermal Growth Factor
Receptor; PARP-1 Inhibitors: Poly-(ADP-Ribose) Poly-
merases 1 Inhibitors; TMA: Tissue Microarray.
Copyright © 2012 SciRes. JCT