2013 International Conference on Signal Processing, Image Processing and Pattern Recognition [ICSIPRI

Development of Electrochemical Biosensor for

Breast Cancer Detection Using Gold
Nanoparticle Doped CA 15-3 Antibody And
Antigen Interaction
1 2 3
P. Georgia Chris Selwyna , Priyadarshini Ravandhu Loganathan , K. Haseena Begam
Department of Biomedical Engineering
I georgias1914@gmail.com
2Idarshinipriya@gmail.com
3hasina456@gmail.com
PSG College of Technology
Coimbatore, India.

Abstract- The diagnosis of breast cancer in the current cancer either begins in the cells of the lobules, which are
scenario is by Mammogram, MRI and its spectroscopy and the milk-producing glands, or the ducts, the passages that
breast biopsy test. All these detection techniques are very drain milk from the lobules to the nipple. Less commonly,
costly, involve surgical techniques, time-consuming and
breast cancer can begin in the stromal tissues, which
results are sometimes negative. Hence, there is a need to
include the fatty and fibrous connective tissues of the
develop a cost effective technique that can qualitatively
and/or quantitatively detect the breast cancer at an early
breast. Over time, cancer cells can invade nearby healthy
stage. Hence the development of a highly sensitive sensor breast tissue and make their way into the underarm lymph
system for monitoring the breast cancer is required so that nodes, small organs that filter out foreign substances in
the patient can remain alert for the risk of getting breast the body. If cancer cells get into the lymph nodes, they
cancer and the level of re-occurrence. This project aims to then have a pathway into other parts of the body. The
develop a label-free electrochemical immunosensor based on breast cancer's stage refers to how far the cancer cells
the CA 15-3 level in serum and the cancer marker have spread beyond the original tumor.
interaction with specific antibody coated with gold
Breast cancer is always caused by a genetic
nanoparticle. These nanoparticles exerts higher surface area
abnormality (a "mistake" in the genetic material).
for enhanced reaction site and higher electrical conductivity
that can provide detectable signals for the sensor which can
However, only 5-10% of cancers are due to an
be read using a electrochemical impedance spectroscopy abnormality inherited from your mother or father. About
(EIS) and potentiostat. 90% of breast cancers are due to genetic abnormalities
that happen as a result of the aging process and the "wear
Keywords- Biosensor, Electrochemical, CA 15-3, Breast and tear" of Iife in general.
Cancer detection

I. INTRODUCTlON
11. CURRENT SCENARIO

Breast cancer is the most common type of cancer
among women worldwide. "Global Breast Cancer
Statistics" has reported that breast cancer is by far the
most common form of cancer for women, comprising
23% of all female cancers [1]. There were 1.15 million
cases of breast cancer in 2002 [2]. Approximately 10% of
newly diagnosed breast cancer patients have a locally
advanced or metastatic disease. Depending on the initial
stage of breast cancers and follow-up treatment strategies
used [3], upto 85% of patients with early diagnosed breast
cancer later develop recurrent or metastatic disease [4].
The term "breast cancer" refers to a malignant tumor
that has developed from cells in the breast. Usually breast
Cancer diagnosis and treatment are of great interest
due to the widespread occurrence of the diseases, high
death rate, and recurrence after treatment. According to
the National Vital Statistics Reports, from 2002 to 2006
the rate of incidence (per 100,000 persons) of cancer in
White people was 470.6, in Black people 493.6, in Asians
311.1, and Hispanics 350.6, indicating that cancer is
wide- spread among all races. Lung cancer, breast cancer
and prostate cancer were the three leading causes of death
in the US, claiming over 227,900 Iives in 2007 alone,
according to the National Cancer Institute. Cancer is also
greatly feared due to recurrences, as although treatable,

978-1-4673-4862-1/13/$31.00 ©2013 IEEE

 2013 International Conference on Signal Processing, Image Processing and Pattern Recognition [ICSIPRI
tumors can return after a period of time, even after
chemotherapy, surgery, or radiotherapy.
Survival of a cancer patient depends heavily on early
detection and thus developing technologies applicable for
sensitive and specific methods to detect cancer is an
inevitable task for cancer researchers.
Existing cancer screening methods include:
l.The Papanicolau test for women to detect cervical
cancer
2. Prostate-specific antigen (PSA) level detection in blood
sampIe for men to detect prostate cancer
3. Occult blood detection for colon cancer
4. Endoscopy, CT scans, X-ray, ultrasound imaging and
MRI for various cancer detection.
III. PROBLEM DEFINITION AND SOLUTION
Existing Breast cancer screening methods include:
(1) Magnetic Resonance Imaging
(2) Mammogram
(3) Biopsy
(4) Fine Needle Aspiration Cytology
These traditional diagnostic methods however are not
very powerful methods when it comes to cancer detection
at very early stages [5]. As weil, some of the screening
methods are quite costly and not available for many
people. Mammograms are most accurate for women in
their 50s because breast tissue is denser among younger
women, making it difficult to detect tumors. Pre­
menopausal women who are in a 'risk' category are
advised CT scan and ultrasounds in addition to a
mammogram. Other technologies like biopsy and fine
needle aspiration are also available, but they are done
only in the case of a positive mammography screening as
they confrrm the disease 100 per cent [6]. The efficacy of
methods such as thermography is yet to be proven
c1inically. Therefore, the development of technology that
is specific and reliable for detecting cancers at early
stages and is easily accessible so that it can function as
the first-line guidance is needed.
The solution to the above stated problem is to develop
an early detection technology for cancer marker
(biomarker) i.e., to develop an inrrnunosensor for
biomarker to detect breast cancer. Biomarkers are
biomolecules present in blood, urine, tissue or any other
body fluids. The levels of which gets elevated from
normal when there is an occurrence of cancer. CA 15-3 is
the specific biomarker for breast cancer.
IV. BIOMARKERcA 15-3
Any specific molecular alteration of a cell on the DNA,
RNA, metabolite or protein level may be referred to as a
molecular biomarker. From a practical point of view, the
biomarker would specifically and sensitively reflect a
disease state and could be used for diagnosis as weil as
2
for disease monitoring during and following therapy [7].
A biomarker is an indicator of a biological state of
disease. It is characteristic of a specific state and therefore
can be used as a marker for a target disease. These
biomarkers can be used to study cellular processes, and
monitor or recognize disruption or alterations in the
cellular processes of cancer cells. These results could
provide us with information on the underlying mechanism
of the initiation of a disease, the process of aggravation,
and ultimately provide a method to diagnose and treat the
disease with appropriate measures at desired stage.
Biomarkers, Specifically cancer biomarkers, are an
indication of cancer and by detecting them the existence
of that specific cancer can be verified.
The most frequently used and known biomarker for
breast cancer is Her2 [8]; this oncoprotein is cell­
membrane bound but its extracellular domain is shed into
circulation making it a potent biomarker used to monitor
the response to treatment, and to detect recurrences in
patients with diagnosed breast carcinoma [9,10].
However, this biomarker is only expressed in 20% of a11
breast tumors [11]; therefore additional breast cancer
markers are rigorously being explored [12]. Mucins are
promising candidates [13] in particular, CA 15-3, a
MUCI Tumor Marker; this cancer antigen is
overexpressed in >90% of breast carcinomas and
metastases [14,15].
The mucin protein product encoded by the MUCI gene
contains approximately 50% carbohydrate by weight with
a relative molecular mass of 400 kDa. This cell surface
mucin transmembrane glycoprotein, is expressed at the
apical surface of most epithelia (e.g. mammary gland,
female reproductive tract, stomach, etc.) in nor- mal tissue
[17]. It is comprised of three structural do- mains: a large
and heavily O-glycosylated extracellular segment (exo­
domain), a hydrophobic type 1 transmembrane region,
and a short cytoplasmic tail domain in- volved in several
signaling processes [18]. However, in cancerous tissue,
this MUC-l biomarker expression can be detected on the
entire cell membrane due to transformation and loss of
polarity [19,20]. After transport to the cell membrane, it
undergoes proteolytic cleavage in which the soluble form
of the large ectodomain is released into circulation [21].
The tumor marker antigen CAI5-3, which corresponds to
an immuno dominant epitope in the extracellular portion
of the membrane bound mucin MUCl, is shed into the
bloodstream. An increase in the serum CA 15-3 shed
ectodomain is associated with progression of carcinoma
in patients diagnosed with breast cancer [16]. Levels of
CA 15-3 measured greater than 35 U/mL are indicative of
the potential progression and recurrence of breast cancer
[22,23] for an iterative PCA (Principal Component
Analysis). The principal component values obtained from
the iterative PCA are used for c1ustering users. For the
purpose of c1ustering, the proposed method employs the
 2013 International Conference on Signal Processing, Image Processing and Pattern Recognition [ICSIPRI 3

RRC method as weIl as the K-means clustering algorithm of the solution. At low pH, most proteins and peptides
for comparison. will be positively charged, while they are negatively
10000
charged at high pH.
n : 100 n : 32 n: 3S n: 15 n : 39 n: 1>5 Fig 1 The gold nanoparticles and the antibody tagged with
gold nanoparticles are confrrmed using UV Spectroscopy,
: . :. :
AFM and TEM images.
'i
1000 '.'
E
� ....
<'> ', '
� I
D. Electrochemical Measurement
� :"1(tI Electrochemical measurements were performed with
'00
.... . . PARSTAT 2273 Advanced Electrochemical System,
,',
' '
, '"" : ... Gamry Echem Analyst. All experiments were carried out
25
.-...
�. .. ... with a conventional three-electrode system comprising a
..: :.:
platinwn wire as auxiliary electrode, a saturated calomel
CONTAOLS TI NO 121'<0 T3·.�O N ... M ...
electrode (SCE) as reference and the working electrode
The above figure shows the concentrations for controls with its area of O.314. To study the conductivity change in
and TNM (Tumor size, Nodal involvement and Presence the AuNP and Ab tagged AuNP solution, the prepared
of Metastasis) classes of breast cancer patients (Pons­ AuNP solution, the Ab tagged AuNP solution is added to
Anicet, et al., 1987). the electrochemical cell and the impedance and CV
measurements are taken at room temperature.
V. MATERIALS AND METHODOLOGY The current vs. voltage measurements were taken using
Keithly 2420 Electrometer. Screen Printed Carbon
A. Materials Electrode was used as the immunosensor. 10f.11 of the
sampie was added to the working area of the SPCE. The
CA 15:3 monoclonal antibody (catalog#: 69249) was
purchased from MP Biomedicals (USA), CA current vs. voltage measurement was selected in the
15:3(catalog#: 771003) Tumor marker antigen was system using Lab tracer 2.0 Sourcemeter Integration
purchased from MP Biomedicals (USA). Screen Printed software (Keithly Intruments, Inc) and the voltage of -5V
to 5V was applied to the working and the reference
Carbon Electrode was purchased from SINSIL
electrode and the graph was plotted between the current
International (USA). All other reagents were of analytical
and voltage. This gave the information about the
grade. Milli pore water was used for all experiments.
conductivity of the AuNP and the antibody tagged AuNP.

B. Preparation OfGold Nanoparticles (AuNP)

The AuNP was synthesized in the size of 3-20nm
according to citrate reduction process .Initially to 19.5ml
of water, 500f.11 of H[AuCI4](lOmM) was added while it is
continuously stirred in ice-cold condition. To the stirring
solution 0.003g of trisodium citrate was added and left for
15 minutes. 0.6ml of NaBH4 (O.IM) was added to the
solution and left for 2hrs continuous stirring. All
glassware used in this procedure was cleaned in freshly
prepared 1:3 HN03-HCI and then rinsed thoroughly in
doubly distilled water. The maximum adsorption of the
synthesized colloidal gold in UV-vis spectra was at 520
nm and the solution was stored in a refrigerator with a
dark-colored glass bottle before use.
C. Antibody Tagged With Gold Nanoparticle:
Gold nanoparticles are physically flexible and suitable
for immobilization of proteins without losing their
conformation or biological activity.Proteins and peptides
acquire their charge mainly through ionization of acidic
and basic amino acids, such as Glu and Lys, respectively.
Therefore the charge of these molecules is highly
dependent on the molecular isoelectric point and the pH
E. Testing With Antigen:
Immunoassay was done for the different concentrations
of the CA 5:3 antigen and the conductivity was found.
The CA 15:3 antigen was predilutted into different
concentration such as 5, 25, 50, and 75 U/ml in different
vials containing Iml each. For the same concentration of
the CA 15:3 monoclonal antibody the different response
for the various CA 15:3 antigen levels was found. 100f.11
of CA 15:3 antibody tagged AuNP was mixed with the 5,
25, 50 and 75 U/ml respectively and they were mixed
thoroughly in vortex. The mixer was remained
undisturbed for 30 minutes in room temperature. 10 f.1l of
each fluid was taken and placed on the working part of
the Screen Printed Carbon Electrode. A voltage of -5 to
5V was applied to the electrode and the corresponding
current was measured.
VI. RESULTS AND DISCUSSION
All the above experiments were done several times till
the appropriate result was obtained. The following are the
results obtained.The fig.2 indicates the UV spectroscopic
image of diluted AuNP recorded using T90+ UV/VIS
Spectrometer (PG Instruments, Ud). The solution showed
 2013 International Conference on Signal Processing, Image Processing and Pattern Recognition [ICSIPRl 4

a broad Surface Plasmon Resonance at around 520 nm Fig 6 TEM Image-AuNP Tagged CA 15-3 Ab
indicating particles of nanodimension. The peak at the
520nm shows the wavelength of AuNP, the absorbance of
the UV is highest at 520nm indicating the presence of
AuNP.
Fig 2: UV Spectrum Of AuNP

Gofd nanoparticle spectrum

L ____
----
----- Fig 7. 2D, 3DAFM Images Of AuNP+CA 15-3 Ab

200 300 400 500 600 700 800

Wavelength in nm

Fig 3 TEM Images Of Au NP

Fig 6 shows a transmission electron micrograph(TEM)

of the CA 15:3 tagged gold nanoparticles. The gold
nanoparticles are embedded on to the matrix of antibody
indicating the binding conjugation of gold nanoparticle on
to the antibody and also the stability of the gold
nanoparticle in the solution. AFM sampies were prepared
on the glass side Fig 7. shows the image AFM image of
the CA 15:3 tagged gold nanoparticles. The yellow spots
indicate the presence of the CA 15:3 tagged gold
nanoparticles. The particles are scanned between the area
Fig 4. EDS Image With AuNP
of 51lm. The antigen-antibody conjugation has been
C Spectrum
150 Au
determined by using a biomimetic surface (based on red
blood cells) sandwiched between gold nanoparticles
deposited on a carbon electrode surface and gold
100
nanoparticles present on the cell surface. These gold
l'Iu
Au nanoparticles have been functionalized with mammary
50 cancer 15-3 antibody (anti-CAI5-3). In the presence of
Au

0
0
�w 2 4
�ilJl
6 8 10 12
Au
_0'\.

14 16 18 20
the antigen, the amperometric response decreased with the
augmentation of CA15-3 sampie concentration.
ull Seale 167 cts Cursor 20.104 (0 cts) kev
A. ELECTROCHEM1CAL MEASUREMENT
Fig 5. 2DAnd 3DAFM Images Of AuNP 1. 1mpedance measurement
The impedance spectra include a semicircle part
at high frequencies, corresponding to the electron
transfer limited process and a linear part at lower
frequencies, resulting from the diffusion limiting step
 2013 International Conference on Signal Processing, Image Processing and Pattern Recognition [ICSIPRI
of the electrochemical process. The semicircle
diameter equals to the electron transfer resistance
(Rct), which indicates the blocking behavior of the
electrode surface for the redox couple and therefore
can be used as a signal for characterizing the
modification for each step. The Nyquist spectrum
obtained for AuNP shows an almost straight line
(Fig.7) indicating the deposition of gold colloidal
nanoparticles on insulating electrode forms a
conductive layer which reduces charge transfer
resistance and consequently the diameter of the
semicircle drastically decreases. The resistance lies in
the value of 2k ohm shows high resistance. After
immobilization of antibody, the interfacial electron
transfer becomes enhanced, leading to a decrease in
the electron transfer resistance (Fig. 8) with the value
of nearby 100 ohm. This shows the CA 15:3 has
bound with the AuNP that ease in the electron
transfer between the AuNP and CA 15:3.
90000 ,------,
80000
70000
60000
50000
40000
30000
20000
10000
-10000 '-------'
20000 40000 60000 80000 100000 120000
Zre (ohm:$)
tr.. (1tlo)
Fig 8. Impedance Spectroscopy of AuNP,AuNP
tagged with CA 15-3 Ab
2. Cyclic Voltammetry (CV)
The CV for AuNP is shown in Fig 9 indicating the god
current response at miroampere. The results obtained
from EIS measurements were in a good agreement with
the results extracted from cyclic voltammograms (Fig 10)
5
and confirms the successful immobilization of CA 15:3
antibody on the surface of AuNP. The green curve shows
CV of CA 15:3 antibody alone which shows very
minimum current response of nearly zero ampere and the
red curve shows the CA 15:3 tagged AuNP which
indicates the increase in current value and a prominent
redox reaction occurring at the tagged moleeule.
3.0E-06,-----"""1 ·
-
2.0E-06
IOE-06
! OOE+OO
u
-IOE-06
-20E-06
-3.0E-06i'------'
-0.2 -0.1 00 0.1 0.2 0.3
PolEntial (V)
Fig 9. CV of AuNP
Figl0. CV of AuNP tagged with CA 15-3 Ab
3. Current Vs Voltage (I Vs V)
The current vs. voltage measurements were taken using
Keithly 2420 Electrometer. In this Screen Printed Carbon
Electrode was used as the amperometric biosensor where
a constant voltage was applied across the electrodes
producing the current response. These methods are able to
detect a change in capacitance and/or resistance of the
electrode induced by binding of protein.IOf.11 of the
sampie was added to the working area of the SPCE. The
current vs. voltage measurement was selected in the
system using Lab tracer 2.0 Sourcemeter Integration
software (Keithly Intruments, Inc) and the voltage of -5V
to 5V was applied to the working and the reference
electrode and the graph was plotted between the current
and voltage. The contact of the solution with the electrode
surface is through the physical adsorption. It is generally
based on interactions such as van der waals forces and
 2013 International Conference on Signal Processing, Image Processing and Pattern Recognition [ICSIPRl 6

electrostatic interactions between the Ab/Ag and the found at the bottom of each vial indicating that the
transducer. It is especially common on hydrophobic antigen and antibody have reacted. 10111 of the sampie
polymer surfaces. The main advantages of this mode of was placed on the working area of the screen printed
immobilization are its rapidity and simplicity, while its carbon electrode and -5 to +5V was applied to the
main drawbacks are random orientation and weak electrode and the current vs. voltage response was plotted.
attachment. The Fig 13 shows the reactivity of CA 15:3 antibody
Fig 11 explains the current vs. voltage response of the tagged AuNP to the CA 15:3 antigen. As the curve infers,
AuNP suspended in water. A constant voltage of -5 to the successive increase in the current as the antigen
+5V was applied to the electrode at the room temperature. concentration increases from 5 to 75 V/mI. Practically no
The current response recorded was found to be in remarkable decrease was observed in current response
microampere. After the repetition of the measurement after fIve measurements.
same results were obtained which shows the stability of
the particles and the screen printed carbon electrode.
-unitS
0006 -unit25
unitSO
-unit75
0 004
.... lO .OOCII!�

0002
O

Si
-'
O,OODI!-t-D

� ·IOO.OOOI!!�
0000
a:
E
�
C .. 002
�
B
5OO , �� � ,,: O' :'. Cü.�'·O: �i.m:ö · ·· O:OCJ:i....'o'· ·'1.:Wi.:o··'··:
40 4 000i ...·o· -. ·6:.m .. 004
Voltltgf!_l

.. 006

.. 008
.. .. ·2
Fig 11.1 Vs V of AuNP voltage(v)

Fig 12 shows the current vs. voltage response of the

CA 15:3 tagged AuNP. . A constant voltage of -5 to +5V
was applied to the electrode at the room temperature. The
current obtained was in milliampere. Shows a drastic
increase in the current from microampere to milliampere
confIrms that the CA 15:3 conjugated with the AuNP.
These results confIrms with the output of Impedance and
CV taken in EIS.
B
' · �� ..: O·' ':'
4 ,m:.o'-'·· :i0
, Cä..:.o····'o:.xu:'+O'·,..··2:000i+·o···· ':
Voltage_l
.I"I"�
.t.Jpj . ..:j
I i---T.=--i:::: •.
-0.007682
Fig 12. 1 Vs V of AuNP tagged CA 15-3 Ab
4. Testing with Antigen (Ag)
The CA 15:3 breast tumour marker antigen was diluted
into different units such as 5 V/mi, 25 V/mi, 50 V/mI, 75
V/mI. The reactivity of the CA 15:3 antibody with the
different concentrations of antigen was studied. To the
different concentrations( 5, 25, 50, 75 V/mi) constant
amount of CA 15:3 tagged AuNP solution was added.
After the mixing the solution in vortex and incubation
after 30 minutes at room temperature, precipitates were
Fig 13. 1 Vs V response for different CA 15-3 Ag
Concentration
VII. CONCLUSI0N
This work describes a new immunosensor based on
immobilization of CA 15:3 antibody tagged colloidal gold
nanopartic\es on Screen Printed Carbon Electrode. CA
15:3 concentration in sampie solution can be detected
based on the current change before and after the antigen­
antibody reaction. The immunosensor permits a reliable
determination of CA 15:3 of 5-75 V/mI. Colloidal gold
nanoparticles and carbon interface of screen printed
carbon electrode could effIciently immobilize antibody
and could be used in the preparation of other
4 m'''O·····6:COOE
amperometric immunosensors for the detection of
important antigens. Therefore, this method has a good
.+ •. ' 'tj>'ll!l
o=
o_
:;"""'1 +
.+
.,.1
".1
application perspective for continuous determination of
CA 15:3.
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