TUGASAN BIOTEKNOLOGI FARMASI

BREAST CANCER BIOMARKERS

KELOMPOK 1
FARMASI KPBI 2014
DOSEN: IBU YUNI ELSA HADISAPUTRI

NAMA NPM BIOMARKER

SHARMILA KARIPAYA 260110142001 EZH2

MUHAMAD IQBAL 260110142002 EZH2

CYNTHIA SANTIAGO 260110142003 CA 27.29

MANDEEP K. 260110142004 CA 27.29

DHILLON

AIDIL NUR MALAYA 260110142005 ESTROGEN RECEPTOR

NUR LIYANA 260110142006 ESTROGEN RECEPTOR

ABDULLAH AZIM 260110142007 uPA and PAI-1

NOR IZZATI 260110142008 uPA and PAI-1

MUHAMAD AFIQ 260110142009 PHOSPHOCHOLINE

SANJIV MENON 260110142010 PHOSPHOCHOLINE

NUR FAZLIANA 260110142011 CEA and CA15-3

NUR SYAHIRAH 260110142012 CEA and CA15-3

FALKUTAS FARMASI
UNIVERSITAS PADJADJARAN JATINANGOR
2017

Journal Title: EZH2 is a marker of aggressive breast cancer & promotes neoplastic
transformation of breast epithelial cells.
Published online by Proceedings of the National Academy of Sciences of the United
State of America(Proc Natl Acad USA) by CG Kleer 2003.

Marker Name: Enhancer of Zeste homolog 2(EZH2)

REVIEW ABOUT MARKER

EZH2 is a histone- lysine N- methyltransferase enzyme encoded by EZH2 gene which

takes place in DNA methylation & eventually in transcriptional repression. Is efficient
enzymatic component of the Polycomb Repressive Complex 2 which is in charge for
healthy embryonic growth thru epigenetic maintenance of genes responsible for
regulating development & differentiation. Mutation or over expression of EZH2 leads
to various forms of cancer. It restricts genes responsible for suppressing tumor
development and by blocking EZH2 action may slow tumor enlargement. EZH2 has
been embattled for inhibition because it is upholding up in multiple cancers including,
but not limited to, breast, prostate, melanoma and bladder cancer.

REVIEW ABOUT JOURNAL

There are several methods used in the jurnal for study of the breast tissue

Selection of Patients and Tissue Microarray Development

Breast tissues for tissue microarray construction were obtained from the Surgical
Pathology files at the University of Michigan with Institutional Review Board
approval. A total of 280 cases (n = 917 tissue microarray samples) were reviewed by
the study pathologist (C.G.K.) and arrayed in three high-density tissue microarrays, as
described (21, 22)

Immunohistochemical Studies

Immunohistochemistry was performed on the tissue microarrays (TMAs) by using

standard biotinavidin complex technique and a polyclonal antibody against EZH2
that was previously validated by immunoblot analysis (8). See Supporting
Methods for detailed methodology. The TMAs were immunostained for ER and PR
and for HER-2/neu by using well described and validated procedures (23).

Statistical Analysis
Comparison of the intensity of EZH2 staining between normal breast, hyperplasia,
ductal carcinoma in situ, invasive carcinoma, and metastases was carried out by
calculating the median staining intensity for each case and applying the Wilcoxon
rank test. A P value of <0.05 was considered significant. Overall survival was
calculated from the date of surgical excision of the primary tumor to the date of death.
Patients who died of or with the disease were included in the analysis. For disease-
specific survival, data for patients who died from other causes were censored at the
time of death. Overall survival and disease-specific survival curves were constructed
by the KaplanMeier method. Clinical criteria for treatment failure were local
recurrence and/or the development of metastases.

Univariate analyses of disease-specific survival were performed by using a two-sided

log-rank test to evaluate EZH2 protein expression, age, tumor size, nodal status, stage,
angiolymphatic invasion, ER status, PR status, and HER-2/neu status. To assess the
influence of several variables simultaneously, a multivariable Cox proportional
hazards model of statistically significant covariates was developed by removing
nonsignificant parameters in a step-wise manner. Statistical significance in the Cox
models was determined by Wald's test.

SYBR Green Quantitative Real-Time PCR

We performed SYBR green real-time quantitative PCR analysis on 19 laser-

microdissected frozen breast tissues obtained from the frozen breast tissue bank in
institution with Institutional Review Board approval.

Immunoblot Analysis

Protein extracts were prepared from normal and cancerous breast tissues and standard
immunoblot analysis performed.

Adenovirus Constructs

Adenoviral constructs were generated by in vitro recombination. In brief, the full-

length EZH2 or SET domain deleted EZH2 (EZH2SET) were inserted in an
adenoviral shuttle plasmid [pACCMVpLpA()loxP-SSP]. Viruses were generated by
transfection into the 293-complementation cell line. Virus was propagated in 911 cells
and purified on a CsCl gradient. Multiplicities of infection were calculated, and
purified viruses were stored in 10 mM TrisHCl (pH 7.4)/137 mM NaCl/5 mM KCl/1
mM MgCl2 in 10% glycerol (by volume).

Cell Count

H16N2 were infected with EZH2 adenovirus. Cell counts were estimated by
trypsinizing cells and analysis by Coulter counter at the indicated time points in
triplicate.

Soft Agar Assay. A 0.6% (wt/vol) bottom layer of low melting point agarose in normal
medium was prepared in six-well culture plates. On top, a layer of 0.6% agarose
containing 1 105 stable transfected cells was placed (24). After 25 days, foci were
stained with P-Iodonitrotetrazolium violet and counted.

HDAC Assay. HDAC activity assays were performed according to the manufacturer's
instructions (Biomol, Plymouth Meeting, PA).

Basement Membrane Matrix Invasion Assay.

Cells were infected with vector, EZH2, and EZH2SET adenovirus. Forty-eight hours
after infection, the cells were trypsinized and seeded at equal numbers onto the
basement membrane matrix 24-well culture plates [extracellular membrane (ECM);
Chemicon] in the presence or absence of HDAC inhibitors suberoylanilide
hydroxamic acid (SAHA) (7.5 M) and trichostatin A (TSA) (0.5 M). FBS was
added to the lower chamber to act as a chemoattractant. After 48-h incubation, the
noninvading cells and ECM were removed gently by cotton swab. The cells that are
invaded that are present on the lower side of the chamber were stained, air dried, and
photographed. The invaded cells were counted under the microscope. For colorimetric
assay, the inserts were treated with 150 l of 10% acetic acid, and absorbance was
measured at 560 nm.
Sea Urchin (SU) Embryo Basement Membrane Invasion Assay

H16N2 cells were infected with vector, EZH2, and EZH2SET adenovirus and
trypsinized after 48 h. The infected cells alone or treated with HDAC inhibitors
SAHA (7.5 M) and TSA (0.5 M) and analyzed for invasiveness by using the SU
embryo basement membrane invasion assay (25).

Chick Chorioallantoic Membrane (CAM) Invasion Assay

EZH2- and control virus-infected H16N2 cells were labeled with Fluoresbrite
carboxylated polystyrene nanospheres of 48 nm diameter (Polysciences) as described
(26).

RESULT

From the jurnal, the result was obtained are EZH2 Transcript and Protein Expression
are elevated in breast cancer. On the basis of previous work characterizing EZH2 in
prostate cancer (8), we were interested in determining whether EZH2 is dysregulated
in breast cancer, which, similar to prostate cancer, is steroid hormone regulated. This
was facilitated by group's ongoing efforts to create a cancer microarray metaanalysis
database (see www.ONCOMINE.org) stemming from the initial work in prostate (27).
Of the five publicly available breast cancer gene expression datasets (2832), only the
Perou et al. (28) study had neoplastic and normal breast tissues to make comparisons
between benign and cancer. Interestingly, in this dataset, we found that the EZH2
transcript was overexpressed significantly in invasive breast cancer and metastatic
breast cancer relative to normal (P = 0.002, t test) (28).

To validate these DNA microarray results, we carried out SYBR green quantitative
real-time PCR on 19 laser-capture microdissected normal and invasive breast cancers.
As predicted, levels of EZH2 mRNA were increased an average of 7.5-fold in
invasive carcinomas compared with normal breast epithelial cells (t test, P = 0.0085) .
To confirm that EZH2 is elevated at the protein level in invasive breast cancer, we
analyzed normal breast and breast cancer tissue extracts by immunoblot analysis.
Consistent with the transcript data, invasive breast cancer expressed high levels of
EZH2 protein relative to normal. Importantly, EED, a PcG protein that forms a
complex with EZH2, did not exhibit similar protein dysregulation..
EZH2 mRNA transcript and protein levels are elevated in breast cancer. (a)
Quantitative SYBR green RT-PCR of EZH2 transcript in laser-capture microdissected
normal and breast cancer epithelia. Each sample was performed in duplicate, and a
ratio was calculated.By using the high-density tissue microarrays, we next evaluated
the expression of EZH2 protein in a wide range of breast tissues (280 patients, n =
917 samples) to characterize its expression in situ by immunohistochemistry. EZH2
protein expression was observed primarily in the nucleus, as reported previously (33).
Invasive breast cancer that expressed high levels of EZH2 (scores 34, EZH2+) and
those that expressed low levels of EZH2 (scores 12, EZH2) were readily apparent.
There was a remarkable staining difference between tumor cells that form
intravascular emboli and adjacent normal breast epithelia. Consistent with mRNA
transcript data, EZH2 protein levels were elevated in invasive carcinoma relative to
normal or atypical hyperplasia. As in the case of metastatic prostate cancer (8), breast
cancer metastases expressed high levels of EZH2. Median EZH2 staining intensities
of normal, atypical hyperplasia, ductal carcinoma in situ (DCIS), invasive carcinoma,
and metastases were 1.47 (SE 0.61), 2 (SE 0), 2.38 (SE 0.52), 2.74 (SE 0.99), and
3.09 (SE 1.04), respectively. Interestingly, increased EZH2 protein and transcript were
already present in DCIS, a precursor of invasive carcinoma.
 Reference

Celina G. Kleer, Qi Cao, Sooryanarayana Varambally, Ronglai Shen, Ichiro Ota, Scott
A. Tomlins, Debashis Ghosh, Richard G. A. B. Sewalt, Arie P. Otte, Daniel F. Hayes,
Michael S. Sabel, Donna Livant, Stephen J. Weiss, Mark A. Rubin, and Arul M.
Chinnaiyan.2003. EZH2 Is A Marker of Aggressive Breast Cancer and Promotes
Neoplastic Transformation of Breast Epithelial Cells. Medical Sciences (available
online https://www.ncbi.nlm.nih.gov/pmc/articles/ PMC208805. Extracted on April
30.2017)

LAMPIRAN
Journal Title: Prevalence of CA 27.29 in Primary Breast Cancer Patients before the
Start of Systemic Treatment

Marker Name: CA 27.29

REVIEW ABOUT MARKER

Description

Cancer antigen 27-29 (or CA 27-29) is an epitope of a large transmembrane mucin

glycoprotein named MUC1 that is expressed on the cell surfaces of most glandular
epithelia. This protein, also known as polymorphic epithelial mucin or epithelial
membrane antigen, has a large extracellular region, a transmembrane sequence, and a
cytosolic domain. CA 27-29 represents a sequence of mucins on the extracellular
region of this glycoprotein. Physiologically, the MUC1 protein may be involved in
protection, lubrication, and cell adhesion by decreasing the degree of cell to extra-
cellular matrix and cell to cell interactions. The MUC1 protein and its overexpression
may be causally related to cancer invasion and metastasis.

The MUC1 protein is often overexpressed and aberrantly glycosylated on its

extracellular surface in malignant granular cells. In fact, the carbohydrate epitopes on
MUC1 in breast cancer cells may be antigenically different than those in normal
breast. As tumor cells shed the MUC1 protein into the bloodstream, CA 27-29 is
recognized by a single monoclonal antibody, which recognizes a 20 amino-acid
sequence in the mucin core. This is in contrast to the CA 15-3 assay, which utilizes
two monoclonal antibodies. However, there is overlap, in part, with the epitopes that
are recognized by both assays. Early-stage breast cancers have a low incidence of
elevated CA 27-29 and lower absolute levels, while higher-stage breast cancers have a
higher incidence of elevated levels and higher absolute levels. Since all breast cancers
do not express the CA 27-29 antigen, this assay cannot detect all breast cancers.

The CA 15-3 assay can be used instead of the CA 27-29 assay. Although CA 27-29
may be more sensitive for breast cancer, its indications and limitations parallel those
of CA 15-3.
Indications/Applications

CA 27-29 for screening, diagnosing, or staging breast cancer

Data is currently insufficient to recommend the use of CA 27-29 for screening,

diagnosing, or staging breast cancer. CA 27-29 is infrequently elevated in early-stage
breast cancers or is completely absent from other breast cancers, making it difficult to
detect early-stage cancers or those tumors that do not express this antigen.
Interestingly, the presence of this tumor marker in early-stage breast cancer may
predict worse outcomes, but its implication on management of early-stage breast
cancers remains unclear. On the other hand, high levels of CA 27-29 can indicate
metastatic disease, but evidence is insufficient to incorporate these marker levels into
the staging system. Furthermore, since both benign and malignant conditions other
than breast cancer can cause elevation in marker levels (Table 1), this assay cannot be
reliably used to screen or diagnose breast cancer.

CA 27-29 for detection of recurrent disease after treatment of primary breast cancer

Currently data are insufficient to recommend the use of CA 27-29 to monitor

recurrence of breast cancer after primary and/or adjuvant treatment of a primary
breast cancer. Several studies have shown that an increase in CA 27-29 may predict
recurrence approximately 5-6 months prior to the onset of signs/symptoms or
positivity of other tests. However, no randomized clinical trials show that early
detection and treatment of these asymptomatic recurrences improve overall survival,
disease-free survival, and quality of life.

Reference Range

Cancer antigen 27-29 (CA 27-29) is used to predict early recurrence of disease in
women with treated carcinoma of the breast.The reference range of serum CA 27-29
is less than 38 U/mL. The upper limit of the range may vary depending on the
laboratory and testing kit used for the test. Values obtained with different assay kits,
methods, or laboratories cannot be used interchangeably.
Interpretation

Elevated CA 27-29 levels can be seen in healthy individuals, in benign conditions,

and in other malignant conditions (see below). [1] CA 27-29 levels tend to remain
relatively stable over time in benign conditions. Thus, an elevated level needs to be
interpreted within the context of the patients history, physical examination, diagnostic
imaging, and laboratory workup findings.

Benign causes of elevated CA 27-29 levels are as follows:

Chronic hepatitis

Liver cirrhosis

Tuberculosis

Sarcoidosis

Benign breast disease

Pelvic inflammatory disease

Endometriosis

Ovarian cysts

Systemic lupus erythematosus

Lactation and pregnancy

Malignant causes of elevated CA 27-29 levels are as follows:

Breast cancer

Small cell lung cancer

Non-small cell lung cancer

Liver cancer

Colon cancer
Gastric cancer

Ovarian cancer

Uterine cancer

Endometrial cancer

Prostate cancer

Pancreatic cancer

Generally, the higher the CA 27-29 level, the more advanced the breast cancer or
larger the tumor burden. If the tumor produces CA 27-29, then tumor marker levels
tend to increase as the tumor grows. The highest levels may be seen in metastatic
breast cancer, particularly with metastases to the liver or the bones. However, CA 27-
29 can be low or absent in all of these settings since not all breast cancers produce CA
27-29, or it may be too early in the disease process to detect CA 27-29 levels. Normal
levels, therefore, do not ensure absence of localized or metastatic disease.

CA 27-29 levels, in conjunction with the history, physical examination, and diagnostic
imaging findings, are typically used to monitor treatment response of metastatic breast
cancer. A decrease in CA 27-29 levels can indicate tumor response or regression of
disease. Stable or increasing CA 27-29 levels, despite adequate treatment, can indicate
tumor non responsiveness or tumor recurrence.

CA 27-29 levels can also be serially obtained after treatment of metastatic breast
cancer. In the absence of measurable disease, an increase in CA 27-29 levels could
indicate treatment failure. A transient rise in CA 27-29 levels can occur during the
first 4-6 weeks of starting therapy; however, this does not usually correlate with
disease progression and should be interpreted with caution.
Collection and Panels

Collection:

Collection details are as follows:

Specimen: Serum

Container: Red-top tube (see the first image below), tiger-top tube (see the second
image below), or gold-top tube (see the third image below). Discuss collection
methods with your laboratory prior to collecting the specimen.

Red-top tube

Tiger-top tube

Gold-top tube
Collection method: Venipuncture

Panels

Panel details are as follows:

Serum CA 27-29 is not part of a panel. CA 27-29 measurement may also be ordered
with estrogen receptor, progesterone receptor, HER2/neu, or other genetic expression
tests. Consideration may be given to ordering multiple tumor markers because of the
heterogeneity in cell composition of each tumor. Other tumor markers to follow breast
cancer include CEA, CA 15-3, HER2 extracellular domain, and CA 125.

CA 27-29 measures an overlapping antigen as the cancer antigen 15-3 (CA 15-3)
assay. One or the other tumor marker is ordered, typically not both.

CA 27-29 for clinical management of metastatic breast cancer

The overall sensitivity and specificity of CA 27-29 is low when this assay is used in
all patients. However, sensitivity and specificity are considerably higher in advanced
(metastatic) or recurrent disease. CA 27-29 levels may increase in higher-stage breast
cancers and larger tumors, which is consistent with its putative role in anti-adhesion,
cancer cell invasion, and metastases.

CA 27-29 can be used in conjunction with the history, physical examination, and
diagnostic imaging to monitor therapeutic response of metastatic breast cancer. CA
27-29, however, should not be used alone to monitor treatment response in this
setting. Additionally, if no other indication of measurable disease (ie, by history,
physical exam, or imaging) are present, an elevation in CA 27-29 can otherwise
signify treatment failure. However, false rises in CA 27-29 may occur in the first 4-6
weeks of therapy, and caution should be used when interpreting these results. [5]

Considerations

Breast cancer is one of several conditions that may cause elevated levels of CA 27-29
(Table 1), and not all breast cancers cause an elevation in marker levels. Thus, a rise
in CA 27-29 levels could be due to conditions other than breast cancer, making
screening and diagnosis difficult.
Early-stage breast cancers have a low incidence of CA 27-29, which confers a low
sensitivity in this population of patients. Although high levels of CA 27-29 can
indicate metastatic disease, evidence is currently insufficient to incorporate CA 27-29
levels into the staging system.

Early recurrence of disease can be predicted by an average of 5-6 months prior to

other signs or symptoms. However, the benefit of this post-treatment monitoring is
controversial, since no studies have shown that detection of early recurrence leads to
more effective therapy and an improvement in patient outcome. The cost-
effectiveness of early treatment and its impact on toxicity also remain unknown.

Low sensitivity in early-stage breast cancer, low overall specificity, and lack of
effective treatment for recurrences are important limitations of the CA 27-29 assay.
[14] Prospective, randomized trials will be needed to further clarify the clinical utility
and impact on patient outcome of CA 27-29.

REVIEW OF JOURNAL

A few trials demonstrate that tumor markers at essential analysis of malignancy have
prognostic pertinence and can foresee scattering of the sickness. While MUC-1
markers are often used to screen treatment viability in metastatic bosom growth, their
part at essential finding or amid follow-up stays hazy. This translational research
extend inside the SUCCESS trial assesses the part of the tumor marker CA 27.29
previously, then after the fact adjuvant chemotherapy, and following two and
afterward five years in patients with early bosom growth. Patients and Methods: The
SUCCESS trial thought about FEC (500/100/500)- docetaxel (100) versus FEC
(500/100/500)- docetaxel/gemcitabine (75/2000) and two versus five years of
zoledronate treatment in hub positive and high-chance hub negative patients with
essential bosom disease. CA 27.29 was measured before chemotherapy in 2669
patients with the reagent ST AIA-PACK CA 27.29 for AIA-600II-Analyzer (Tosoh
Bioscience, Belgium). Consequences of CA 27.29 over 31 U/ml were viewed as
positive. Comes about: 7.6% of patients had raised marker levels after the
consummation of essential surgical treatment yet before start of chemotherapy
(n=202, mean 19, territory 3-410 U/ml). No connection between's nodal status
(p=0.55), reviewing (p=0.85), hormonal status (p=0.21), HER2/neu status on the
essential tumor (p=0.58) and CA 27.29 was appeared. Be that as it may, bigger tumor
measure (p=0.02), lobular histology (p<0.0001), more seasoned age (p<0.001) and
postmenopausal hormone status before the begin of treatment (p=0.006) were
altogether connected with higher CA 27.29 levels. Decision: These information
demonstrate a cozy connection between CA 27.29 and tumor mass holding on even a
little while after surgery, additionally distinguish potential puzzling elements that
ought to be considered in deciphering tumor marker comes about.

Additionally follow-up of the SUCCESS trial will clear up whether CA 27.29

measured after surgery however before the begin of systemic treatment is
prognostically important and whether it is a valuable marker for treatment checking in
the adjuvant setting.

Pervasiveness of CA 27.29 inspiration at essential finding. The serum tests were

obtained after essential surgical treatment prompting R0 resection and before the
begin of chemotherapy. Around then point, 7.6% of patients gave lifted marker levels
(n=202). The mean CA 27.29 esteem was 19 U/ml, with a range from 3 U/ml to 410
U/ml). CA 27.29 levels sorted by percentiles are delineated in Figure 1. The middle
age of all patients was 53 years (run 21 to 76 years). The greater part of patients had
little tumors (41.6% T1, 51.5% T2, 5.7% T3, 1.2% T4) and 34.3% were hub negative
(45.5% N1, 14.2% N2, 6.1% N3). By and large, 4.7% gave G1 tumors, 71.4% of
patients were hormone-receptor positive and 25.5% Her2/neu positive on the essential
tumor.

Figure 1. CA 27.29 values after surgical treatment but before the start of systemic
chemotherapy.
Relationship of CA 27.29 at essential analysis with traditional prognostic markers.
Table I outlines the patient attributes at the season of essential conclusion in
connection to CA 27.29 inspiration. Raised CA 27.29 levels did not connect with the
lion's share of tumor attributes at the season of essential conclusion, for example,
nodal status (p=0.55), histopathological evaluating (p=0.85), hormone receptor status
(p=0.21) or Her2/neu status on the essential tumor (p=0.58), nor with the surgical
treatment (p=0.08 for bosom surgery and p=0.31 for axillary treatment) or systemic
chemotherapy randomization (p=0.5). Nonetheless, bigger tumor estimate (p=0.02)
and lobular histological tumor sort (<0.0001), and in addition more established age
(p<0.001) and postmenopausal status before the begin of treatment (p=0.006) were
fundamentally connected with higher CA 27.29 levels.

These information demonstrate a cozy connection between CA 27.29 and the tumor
mass continuing even half a month after essential surgery. Moreover, we distinguished
tumor and patient qualities that ought to be considered in tumor marker
understanding. Longer follow-up inside the SUCCESS trial will give data on the
prognostic importance of CA 27.29 preceding the begin of systemic treatment and
will exhibit the part of this tumor marker in adjuvant chemotherapy and endocrine,
and also bisphosphonate, treatment checking.
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Link Journal:

http://ar.iiarjournals.org/content/30/5/1837.full?cited-by=yes&legid=anticanres;30/5/1837

Journal Title: Tumor Markers of Breast Cancer: New prospectives

Journal of Oncological Sciences

April 2017, Vol.3(1):511, doi:10.1016/j.jons.2017.01.001

Department of Clinical Pharmacy, College of Pharmacy, Taif University, Taif, Saudi

Arabia. Department of Pharmacology, Faculty of Medicine, Tanta University, Tanta,
Egypt. Received 29 August 2016. Revised 24 November 2016. Accepted 8 January
2017. Available online 16 January 2017.

Marker Name: Estrogen Receptors (ER)

INTRODUCTION

A tumor marker is a biomarker that is found in blood, urine or body tissues that can be
elevated by the presence of one or more types of cancer. It is produced either by the
tumor itself or by the host in the response to a tumor. The ideal tumor marker should
be both specific and sensitive to detect small tumors to allow early diagnosis or help
in screening. Few markers are specific for a single tumor. Most markers are produced
by different tumors of the same tissue type. They are present in higher quantities in
cancer tissue or in blood from cancer patients more than in the blood of normal
subjects. Tumor markers are mostly useful in evaluating the progression of the disease
status after initial chemotherapy and radiotherapy to monitor subsequent treatment
strategies.

Breast cancer is the second most common type of cancer after lung cancer (10.4% of
all cancer incidence, both sexes counted) and the fifth most common cause of cancer
death. It is a disease caused by a combination of genetic and environmental factors.
Numerous risk factors that may be associated with breast cancer have been
recognized. Not all breast cancer patients have the same clinical picture. Some factors
increase a woman's risk of breast cancer more than others.

Early detection of breast cancer both primary and recurrent, is of considerable clinical
importance, and it can be used to make treatment decisions while tumor burden is low,
and when patients are most likely to respond to adjuvant therapy. In recent decades,
the serum concentration of tumor markers has been used to detect tumor activity.
Tumor markers provide a minimally invasive cost-effective source of data valuable
for monitoring disease course, determining prognosis, and helping in treatment
planning. An understanding of the individual test characteristics and limitations is
important for optimal use and accurate interpretation of results.The real usefulness of
tumor markers in the management of breast cancer has been questioned because of the
low diagnostic sensitivity for early disease.
The American Society of Clinical Oncology (ASCO) has updated its
recommendations for use of tumor markers in prevention, screening, treatment and
surveillance of breast cancer. 13 categories of breast tumor markers were considered.
The tumor markers that showed evidence of clinical utility and were recommended
for use in practice include CA 15-3, CA 27.29, Carcinoembryonic antigen (CEA),
Estrogen receptor (ER), Progesterone receptor (PR), Human epidermal growth factor
receptor 2 (HER2), Urokinase plasminogen activator (uPA), Plasminogen activator
inhibitor 1 (PAI-1) and multiparameter assays for gene expression.8 However, other
categories are also used in screening of breast cancer but they demonstrated
insufficient evidence support routine use in clinical practice including P53, cathepsin
D, cyclin E and nestin.

ESTROGEN RECEPTOR (ER)

ER is one of the successful tumor markers in breast cancer. The ER has a role in
cellular growth, proliferation and differentiation. In addition to prognostic value, ER
is the most important biologic marker of response to treatment in breast cancer. It is a
member of the family of nuclear steroid receptors and functions as a transcriptional
regulator, which is controlled by the hormone 17p-estradiol estrogen (E2). Hormone
activated estrogen receptors form dimers, and since the two are coexpressed in many
cell types, the receptors may form ERa homodimers or ERp heterodimers. ERa is
localized on human chromosome 6, in contrast to ERp, which i chromosome 14.

Measurement of ER levels in breast tumor tissue is useful as a prognostic indicator

and in determining the probability of hormonal resistant breast cancer. It was
recommended that ER should be measured in every invasive breast cancer as well as
in metastatic lesions if the results would influence the treatment plan.

In both pre- and postmenopausal patients, steroid hormone status should be used to
identify patients most likely to benefit from endocrine therapy such as tamoxifen, and
raloxifene in both the early setting and metastatic disease. Clinically, a positive ER-a
status correlates with favorable prognostic features, including a lower rate of cell
proliferation and histological evidence of tumor differentiation. ER-a status is also
prognostic for the site of gross metastasis. Han et al. (2016) found that ER-a status
predicts late-onset skeletal metastasis in breast cancer patients. However, Beije et al.
(2016) reported that discordances regarding ER status between circulating tumor cells
and the primary tumor occurred frequently but had no prognostic impact in metastatic
breast cancer patients.

The greater the ER content of the tumor, the higher the response rate to endocrine
therapy Women with systemically untreated ER-positive/Progesterone (PR)-positive
tumors have better clinical outcomes compared with women with ER-negative/PR-
negative tumors, confirming the prognostic significance of the receptor-positive
phenotype.

The potential role of ER determination in the management of Carcinoma In-Situ

(CIS), which is a complex group of diseases that diverse outcomes and account for
approximately 20%30% of breast cancer patients, has attracted a particular attention.
As ER negativity is associated with a worse outcome in patients with CIS, it is not an
independent predictor in the context of high nuclear grade and necrosis.

False-positive results of ER assays (ER-positive tumors but no response to endocrine

therapy) are more common than false-negative results. The most frequent explanation
is heterogenicity of tumor with biopsy of a site that is not representative of the other
tumor deposits. In addition to this problem, there exists an evidence that some tumor
cells have functional receptor defects distal to the initial binding steps (e.g., variant
cells are able to bind steroids in the cytoplasm but will not transport the receptor to
the nucleus).

Reference

Kabel, A.M., 2017. Tumor Markers of Breast Cancer: New Prospectives. Journal of
Oncological Sciences, 3(1), pp.5-11.

LAMPIRAN
Urokinase Plasminogen Activator (uPA) and Plasminogen Activator Inhibitor 1
(PAI-1)

Plasminogen activating proteins such as urokinase-type plasminogen activator (uPA),

plasminogen activator inhibitor-1 (PAI-1), and uPA receptor (uPAR) represent reliable
tumor markers. High levels of uPA, PAI-1, and uPAR in tumor tissue usually correlate
with poor prognosis in many types of human cancers, including breast, endometrial,
ovarian, colon, lung, stomach, and renal cancer.
The urokinase plasminogen activator and its inhibitor are proteins found on the
outside of breast cancer tumor cells that help the cancer grow and spread. Urokinase
plasminogen activator (uPA) and plasminogen activator inhibitor type-1 (PAI-1) play
a key role in tumour invasion and metastasis.Testing for these two proteins is done on
breast cancer tissue removed during biopsy or surgery.

uPA is a 53-kDa trypsin-like protease that converts the plasminogen into active
plasmin. In vivo, uPA catalytic activity can be inactivated by several inhibitors,
including PAI-1, PAI-2, and maspin. PAI-1 was thought to be the primary inhibitor of
uPA. In addition to binding to uPA, PAI-1 can also attach itself to the extracellular
matrix protein (EMP) allowing PAI-1 to modulate cellular adhesion and migration.
uPA was proven to be involved in cancer invasion and metastasis. Antibodies and
inhibitors of uPA prevent or reduce metastasis. Prevention of uPA from binding to its
receptors decreases the formation of metastases. It was believed that uPA promoted
cancer dissemination by degrading the ECM, thus allowing cancer invasion and
metastasis. uPA has the ability to stimulate angiogenesis, mitogenesis, and cell
migration and to modulate cell adhesion. Moreover, uPA was shown to prevent
apoptosis which will increase the survival of malignant cells during the metastatic
process, thus increasing the possibility for the establishment of a secondary deposit.
PAI-1 is an inhibitor of uPA that is expected to prevent invasion and metastasis.
Tumor expression of urokinase-type plasminogen activator (PAI-1), and uPA receptor
(uPAR) represent important breast cancer prognostic factors.

Test results are reported as a score:

0.0-0.4 is normal

0.41-2.8 is borderline

2.81-5.7 is high
Doctors may test for levels of the urokinase plasminogen activator protein and its
inhibitor protein in women diagnosed with hormone-receptor-positive, HER2-
negative breast cancers that have not spread to the lymph nodes (node-negative
cancer).

In many cases, cancers with these characteristics are considered to have a low risk of
recurrence (the cancer coming back). Knowing the levels of these two proteins can
help women and their doctors decide whether chemotherapy or other treatments are
needed after surgery.

JOURNAL REVIEW

Title: Markers of the uPA System and Common Prognostic Factors in Breast
Cancer

Study conducted by;

Alessandro Marco Minisini, Dora Fabbro, Carla Di Loreto, Marta Pestrin, Stefania
Russo, Giovanni Gerardo Cardellino, Claudia Andreetta, Giuseppe Damante, and
Fabio Puglisi.

REVIEW JOURNAL

The urokinase plasminogen activator (uPA) system includes uPA and plasminogen
activator inhibitor types 1 (PAI-1) and 2 that mainly act by regulating extracellular
matrix degradation, and it is involved in tumor progression. The 675 4G/5G
polymorphism of the PAI-1 gene regulates PAI-1 activity in serum.

The aimed of this study is to studying the 675 4G/5G polymorphism of the PAI-1
gene and uPA, PAI-1, and cyclooxygenase-2 (COX-2) immunohistochemical
expression in a series of breast cancer cases.

For each patient, tumor samples were reviewed for histo- logic diagnosis, nodal
status, histologic grade, immunohisto- chemical analysis of estrogen receptor (ER),
progesterone receptor (PR), MIB-1, HER-2, uPA, PAI-1, and COX-2. Blood samples
were obtained from all participants to extract genomic DNA for the genotype
determination.

Genomic DNA was extracted from 5 mL of venous blood, and the status of the
polymorphism 675 4G/5G of the PAI-1 gene was evaluated by polymerase chain
reaction (PCR), using an allele-specific PCR procedure. Oligonucleotides used as
primers were as follows: 5'-GTCTGGACACGTGGGGA-3' as the forward primer to
detect the 4G allele; 5'-GTCTGGA- CACGTGGGGG-3' as the forward primer to
detect the 5G allele; and 5'-TTTTCCCCAGGGCTGTCCA-3' as the reverse primer.

The PAI-1 promoter region polymorphism was assessed in 193 patients with breast
cancer: 56 (29.0%) had 4G/4G homozygosity, 52 (26.9%) had 5G/5G homozygosity,
and 85 (44.0%) had the 4G/5G genotype, with frequencies of 0.51 and 0.49 for alleles
4G and 5G, respectively. In a control pop- ulation of 142 unaffected women, 39
(27.5%), 35 (24.6%), and 68 (47.9%) had the 4G/4G, 5G/5G, and 4G/5G genotypes,
respectively. The allelic frequencies in the control population were 0.51 and 0.49 for
the 4G and 5G alleles, respectively. Therefore, in terms of genotype and allele
frequency, no significant difference exists between control subjects and patients.
4G/4G homozygosity was associated with positive nodes at diagnosis (P = .03, 2 test;
odds ratio, 1.63; 95% confidence interval, 1.02-2.62). No statistically significant
association were found between PAI-1 promoter region polymorphism genotypes and
PAI-1 protein expression in tumor or in stroma cells; no association of genotype with
other tumor characteristics was evident.

Median scores of immunohistochemical expression for uPA, PAI-1, and COX-2 were
100, 70, and 100, respectively; 8% of samples were negative for uPA, 9% for PAI-1,
and 3% for COX-2 expressions. Positive correlations were found between uPA and
PAI-1 expression, between uPA and COX-2 expression, and between PAI-1 and COX-
2 expression. Direct correlation between uPA and ER expression (Spearman = 0.2;
P = .03). Absence of uPA expression in cancer cells was associated with negative
hormone receptor status (P < .05; Fisher exact test), higher histologic grade (P < .05;
Fisher exact test), and higher proliferative activity, as determined by MIB-1
immunoreactivity (23.9% vs 8.9%; P = .005; Mann-Whitney U test). In addition,
intense expression of HER-2 (score 3+) was significantly associated with low
expression of uPA (P = .03; Mann-Whitney U test).

Positive expression of uPA in tumor cells was associated with positive expression in
tumor stroma fibroblasts (P < .05; 2 test). Similarly, there was also an association
between tumor cells and fibroblast expression of PAI-1 (P < .05; 2 test). High COX-2
expression was associated with younger age at diagnosis (Spearman = 0.2; P = .03)
and intense HER-2 expression (score 3+ vs < 3+; P = .003; Mann-Whitney U test).

As a conclusion, this study showed a positive correlation between the uPA system and
COX-2 expression. This finding supports previous observations suggesting the
existence of a link between the uPA- and metalloproteinase-mediated tumor invasion
activity and the functional role of COX-2 in the metastatic process. In particular, it
has been demonstrated that COX-2 up-regulates matrix metalloproteinases (MMPs).
In a corneal model, COX-2 increased uPA, MMP-1, and MMP-9 levels. Similarly,
colorectal tumor cells treated with COX-2 inhibitor (NS398) showed a decrease in
uPA messenger RNA and proliferation. COX-2 increased cellular migration and
invasion in breast cancer cells through enhancement of pro- uPA levels.26 Sivula et al.
evaluated COX-2 and MMP-2 expression in a series of breast carcinomas. A
significant association between MMP-2 and COX-2 was found, and high expression
of both markers resulted in decreased disease specific survival. New insights of
mechanisms of tumor invasion and metastasis are eagerly awaited. In breast cancer, a
better understanding of the relationship between expression of the uPA system and
COX-2 is of valuable biologic importance and could translate into new therapeutic
opportunities.

REFERENCES

Evans DM, Sloan Stakleff KD. Control of pulmonary metastases of rat mammary

cancer by inhibition of uPA and COX-2, singly and in combination. Clin Exp

Metastasis. 2004;21:339-346.

Konno H, Baba M, Shoji T, et al. Cyclooxygenase-2 expression correlates with uPAR

levels and is responsible for poor prognosis of colorectal cancer. Clin Exp
 Metastasis. 2002;19:527-534.

Li G, Yang T, Yan J. Cyclooxygenase-2 increased the angiogenic and metastatic

potential of tumor cells. Biochem Biophys Res Commun. 2002;299:886-890.

Singh B, Berry JA, Shoher A, et al. COX-2 overexpression increases motility and

invasion of breast cancer cells. Int J Oncol. 2005;26:1393-1399.

Sivula A, Talvensaari-Mattila A, Lundin J, et al. Association of cyclooxygenase-2 and

matrix metalloproteinase-2 expression in human breast cancer. Breast Cancer Res

Treat. 2005;89;215-220.

Link journal

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REVIEW JURNAL PHOSPHOCHOLINE

In the present study we found that phosphocholine accumulates in breast cancer cells
as a result of enhanced choline transport into the cells and increased intracellular
choline kinase activity, several fold above that of CTP:PCho cytidylyltransferase. We
also showed that these metabolic changes are predominantly caused by the
upregulation of specific choline transporters and choline kinase genes.
Choline transporters were found to be expressed in the normal and cancerous
mammary epithelial cells in the following order: CTL1 OCT2 OCT1 CHT1. Among
these four transporters, CTL1 and OCT1 were found to be expressed at similar levels
in both normal and malignant cells, whereas OCT2 and CHT1 expression was
upregulated in most of the malignant cells. We further demonstrated that the
upregulation in the expression of the choline transporters was in accord with the
increase in choline transport rate in these cells.

Furthermore, the increased transport rates correlated with the rise in phosphocholine
level, indicating the important role played by the transport in determining PCho level.
In a recent study of both spontaneous immortalized human mammary epithelial cells
(MCF-12A), and MDA-MB-231 human breast cancer cells it was found that all
choline transporter genes were similarly expressed.Indeed, we found that unlike in
most of the breast cancer cells, the increase in the expression level of CHT1 and
OCT2 in MDA-MB-231 cells was not significant; however choline transport rate was
significantly faster (by twofold) in these cells when compared with HMEC.

We also showed in-here that both normal and malignant mammary cells express
choline kinase a and choline kinase b. However, the mRNA expression level of
ChoKb was found to be similarly expressed in the normal and 3 of the cancer cell-
lines and increased by less then twofold in ZR-75-1 and SKBR3 cells, whereas the
mRNA expression level of ChoKa was selectively and markedly upregulated in all the
cancer cell-lines. It was previously shown that the 2 rat choline kinases are active
either as homodimers (a/a or b/b) or as a heterodimer (a/b), and that the activity of a/a
is much higher than that of b/b.Thus, the increased mRNA expression of ChoKa in the
malignant human mammary cells could affect choline kinase activity not merely by
the increased expression, but also by increasing the fraction of the more active choline
kinase dimers a/a and a/b.

The association of choline kinase activity with malignant transformation has been
extensively explored in various cancer celllines and tumors. Specific evidence that
choline kinase plays an important role in malignant transformation of the breast was
previously demonstrated by showing that choline kinase is overexpressed in human
breast cancer, with respect to its expression in normal human mammary epithelial
cells. Furthermore, choline kinase was shown to be necessary for growth
factorinduced proliferation in human mammary epithelial cells as well as for
mitogenic response to growth factors.

PHOSPHOCHOLINE AS A BIOMARKER OF BREAST CANCER 1727 note that in

vivo studies of human breast cancer by means of magnetic resonance spectroscopy
showed a much higher incidence (83%) of choline accumulation in breast cancer
lesions.These variations may reflect differences in the sensitivity of the 2 measuring
methods or indicate that there might be other factors, beside choline kinase activity,
that could elevate the composite choline signal detected by MRS. Reduction in CCT
activity could also lead to increased PCho level, though we did not find a common
trend in the activity of CCT in the cancer cells when compared with HMEC.

Generally, all human breast cancer cells studied thus far show higher level of PCho
when compared with HMEC. Therefore, it is reasonable to suggest that increased
PCho reflects malignant transformation of mammary epithelial cells. However, in
order to investigate whether phosphocholine level and the genes associated with its
elevation are related to the staging and grading of breast cancer cells it is necessary to
use rigorous criteria for defining differentiation, invasiveness and matastatic potential,
as well as a large number of different cell-lines (or tumors).

Furthermore, the supply of choline in tumors in vivo is different than in cultured cells
and is much more uneven due to perfusion heterogeneity. Presumably, the
microenvironment conditions could markedly affect the level of PCho and other
choline metabolites. Therefore, our study serves as a basis for understanding the
molecular machinery at the cell level responsible for the accumulation of choline
metabolites in breast cancer under optimal growth conditions. Further studies in 3D
cell systems (spheroids), experimental tumors and eventually in patients are necessary
to find out how the microenvironment affect this cellular process and whether the
level of choline metabolites can predict prognosis.

LAMPIRAN
NAME OF MARKER :
Carcinoembryonic antigen (CEA) and cancer antigen 15-3 (CA15-3)

REVIEW ABOUT MARKER :

Carcinoembryonic antigen (CEA) and cancer antigen 15-3 (CA15-3) levels is used as
prognostic, screening, diagnosis, staging, or routine surveillance in patients with
breast cancer. Though the European Group on Tumor Markers has recommended the
CEA and CA15-3 levels be used for assessing prognosis, the early detection of disease
progression, and treatment monitoring in breast cancer. Thus this study is done to
explore the relationships between preoperative serum CEA, CA15-3 levels and
clinicopathological parameters, as well as the prognostic value of these two serum
biomarkers in breast cancer.

In this retrospective analysis, a total of 432 patients who were treated for stage IIII
invasive breast cancer at The Affiliated Cancer Hospital of Zhengzhou University
were investigated. TNM, PR, ER, HER-2 criteria is cornfirmed using FISH, nuclear
stain cell. The molecular subtypes were classified into four groups as follows:
Luminal A (ER+and/or PR+, HER2-,Ki-67<14%); Luminal B (ER+ and/or PR+,
HER2+ and/or Ki-67 >14%); HER2 positive (ER- and PR-, HER2+); and triple-
negative (ER- and PR-,HER2-). Serum CEA and CA15-3 levels were determined
using an automatic electrochemistry luminescence immunoassay system (ROCHE
E170; Roche, Germany). DFS and OS were estimated using the KaplanMeier
method and compared using the log-rank test. Independent prognostic factors for DFS
and OS were identified by multivariate Coxs proportional hazard analysis. All the
statistical analyses were performed with SPSS 17.0 (SPSS, Chicago, IL, USA)
software.

The result shows that there are significant elevation of serum CEA and Ca15-3 with
patients which, 10.9% and 13.9%. Both serum were correlated with the size of the
primary tumor and axillary lymph node status, larger tumor size, advanced axillary
lymph nodal and TNM stage exhibited higher proportion of elevated serum tumor
markers. The elevation of CEA levels was significantly greater in patients with HER2
positive tumors while CA15-3 elevation was significantly greater in patients with ER
negative tumors. Clinicopathological factors such as age, tumor size, node metastasis,
ER status, HER2 status and molecular subtype also had independent prognostic
power. For prognostic factors for DFS and OS, base on Multivariate Coxs regression
analysis according to tumor size, nodal status, stage, HER2 status, and serum tumor
markers revealed that elevated serum CEA and CA 153 levels were independent.
Combination of two markers shows the best result for normal patients while for
patients with highly alleviated results shows the worst survival. Luminal B also shows
that both CEA and CA 153 levels were independent prognostic factors for DFS and
OS in breast cancer patients.

In conclusion, although there are few research that were contradicting each other,
usage of both serum shows significant result. Elevated preoperative serum tumor
markers could be useful in determining the risk of recurrence and metastasis of breast
cancer after operation, and further analyses, including a prospective study design, are
needed to clarify the utility of these serum biomarkers in treatment decision-making
area for breast cancer.

REFERENCE
Shao Y, Sun X, He Y, Liu C, Liu H (2015) Elevated Levels of Serum Tumor Markers
CEA and CA15-3 Are Prognostic Parameters for Different Molecular Subtypes
of Breast Cancer. 2015; PLOS ONE 10(7): e0133830. doi :
10.1371/journal.pone.0133830

LAMPIRAN