Cancer Biomarkers

Types of Biomarkers
Serological biomarkers are an effective and relatively non-invasive approach for the
early detection, diagnosis, prognosis and management of many types of diseases,
including ovarian cancer. A biomarker is defined as a quantifiable characteristic that is
objectively measured and evaluated as an indicator of a normal biologic process, a
pathogenic process, or a pharmacologic response to a therapeutic intervention. Typically,
they are endogenous molecules that can be measured in bodily fluids or tissues with the
ability to distinguish between disease and normal states. Cancer biomarkers may appear
in different types and forms, including DNA, mRNA, proteins, metabolites, or processes
such as apoptosis, angiogenesis or proliferation (65). Additionally, different functional
subgroups of proteins, such as enzymes, glycoproteins, oncofetal antigens and receptors,
may serve as useful biomarkers. Furthermore, tumor changes such as genetic mutations,
amplifications, translocations and changes in microarray profiles (signatures) may also be
utilized as tumor markers. Tumor markers may be detected in a variety of fluids, tissues
and cell lines as they are often produced by the tumor itself or by other tissues in response
to the presence of cancer or other associated conditions, such as inflammation. By
measuring the levels of such markers through a serological test, tumor markers can be
used for population screening, differential diagnosis in symptomatic patients, and for
clinical staging of cancer. In addition, they may also be used to estimate tumor volume, to
evaluate response to treatment, and to assess recurrence through monitoring or as
prognostic indicators for disease progression (76).
Biomarkers are classified as being diagnostic, prognostic, or predictive. Diagnostic markers
are applied in disease detection and in identifying a given type of cancer in an individual. To
minimize false positive and false negatives rates, diagnostic markers are expected to have
high sensitivity and specificity. Screening markers are a specific type of diagnostic markers
where they are used to examine the general population for a disease (48). Currently, there is
no perfect screening marker for ovarian cancer. Prognostic markers, on the other hand, are
used once the disease state has been established and are applied to determine the
probability of a patient responding to therapy in order to improve the accuracy of medical
prediction and the etiology of the disease following tumor resection. These markers are
expected to predict the likely course of the disease, its recurrence, and influence the type of
therapy provided to the patient. Currently, FIGO staging is the major prognostic factor for
ovarian cancer to identify patient prognosis and treatment for ovarian cancer patients.
Lastly, predictive markers are used to predict the response to a drug before treatment is
initiated. Optimally, it is able to classify individuals as likely responders or non-responders to
a particular treatment. Often, predictive markers arise from array-type experiments that
make it possible to predict clinical outcome from the molecular characteristics of the
patients tumor. Unfortunately, other than definitive diagnosis by biopsy and
histopathology, there is currently no single diagnostic, prognostic, or predictive tumor
marker with acceptable sensitivity and specificity for ovarian cancer.


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The Ideal Tumor Marker

The World Health Organization (WHO) lists specific criteria that a biomarker must
satisfy. According to WHO, a good screening test must meet the following criteria (31):
1. There are significant mortality statistics for the target disease and occurrence in the
population 2. Disease progression should be well characterized 3. Early stage treatment of
the disease should offer improved outcome 4. Public acceptance of the screening test 5.
Availability of effective treatment options for individuals with advanced disease 6.
Suitable treatment and diagnostic facilities 7. Policy outlining who can be subjected to
treatment 8. Cost-effective screening 9. High positive predictive value, negative
predictive value, sensitivity and specificity In addition to the above criteria, an ideal
tumor marker should be measured easily, quickly, reliably and cost-effectively using an
assay with high analytical sensitivity and specificity (48). Its differential expression in a
significant portion of the patient population should be characteristic of the cancer of
interest, and rarely occur for other conditions or for normal patients (41). The ideal
marker should be produced by the tumor cells and enter the circulation in order for it to
be detected by a non-invasive serological test. The marker should be present at low levels
in serum of healthy or benign disease patients and increase significantly in cancer
(preferably in one cancer type). Optimally, an ideal marker is present in detectable (or
higher 20 than normal) quantities at early or preclinical stages and the quantitative levels
of the tumor marker should reflect the tumor burden. The assay for this marker should
demonstrate high diagnostic sensitivity (low false negatives) and high specificity (low
false positives). Current tumor markers for ovarian cancer, such as carbohydrate antigen
125 (CA125), suffer from low diagnostic sensitivity and specificity when used alone.
Consequently, they are used in conjunction with imaging, biopsy and associated
clinicopathological information prior to setting a diagnosis or prognosis.

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Cancer Antigen-125
Currently, the clinically accepted serum marker for ovarian cancer is carbohydrate
antigen 125 (CA125), a high molecular weight mucin (glycoprotein) with unknown
function (177). It was discovered initially by a radioimmunoassay in patients with
advanced ovarian cancer (9). It is expressed by fetal amniotic and coelomic epithelium,
and in adult tissues that are derived from the coelomic (mesothelial cells of the pleura,
pericardium and peritoneum) and Mullerian (tubal, endometrial and endocervial)
epithelia. Normal epithelium of the ovaries does not express CA125 on the surface (83).
While CA125 may be the best ovarian cancer biomarker discovered to date, its utility as a
screening marker is limited due to its high false positive rates. It is elevated in other
malignancies such as uterine, fallopian, colon and gastric cancer (77, 166) as well as in
1% of the normal population, particularly in non-malignant conditions such as pregnancy,
menstruation and endometriosis (9-11, 77, 166). In addition, many prospective studies of
screening have revealed major limitations of CA125 also in its sensitivity (16).
Specifically, the sensitivity of CA125 is more than 90% for women with advanced stage
ovarian cancer, but the 21 sensitivity for stage I ovarian cancer decreases to
approximately 50%. As a result, its clinical use for the early detection of ovarian cancer is
limited (16, 77, 78). Therefore, CA125 is neither sensitive nor specific enough to be used
as a diagnostic biomarker. Serum CA125 levels greater than 35 U/mL are considered
elevated (166). These levels may occur one to two years prior to conventional diagnosis
(11, 44, 45, 166, 182). Screening using CA125 may detect a proportion of ovarian cancer
cases before symptoms arise (68, 78, 79). CA125 has been found to be particularly useful
in detecting early relapse (112). CA125 has also been shown to play a significant role in
prognosis. Some studies have demonstrated that concentrations of CA125 in the serum
decrease with tumor regression, and increase with progression in 74 to 95% of cases (13,
166). In addition, CA125 has been able to predict survival outcomes in women with
CA125 levels greater than 65 U/mL (116). Although CA125 may be promising in its
prognostic value, the current major prognostic factor is the FIGO stage. Other
conventional prognostic markers include variables such as tumor grade, size, histological
subtype, residual tumor after surgery, and patient age. However, ovarian cancer is a
highly heterogeneous disease, thus, cancers with similar clinical profiles may have
different outcomes. Although CA-125 is used currently in clinical settings for diagnosis
and prognosis, its limitations in sensitivity and specificity warrant the development and
identification of novel ovarian cancer biomarkers that would complement CA125 in
facilitating early disease detection, determination of prognosis, and the development of
more individualized and efficient treatment plans for ovarian cancer patients.
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Mechanisms of biomarker elevation in biological fluids

Protein levels are physiologically maintained in biological fluids. In disease states,
proteins may become elevated as a result of the disease by several mechanisms. These
include and are not limited to gene over-expression; angiogenesis, invasion and
destruction of tissue architecture; and finally increased protein secretion and shedding.
First, increased protein quantities may be due to increases in the specific gene or
chromosome copy number (gene amplification), epigenetic modifications such as DNA
methylation, and increased transcriptional activity. Increased transcriptional activity is
often due to the imbalance between gene repressors and activators. Second, tissue
invasion by the tumor may allow direct release of molecules into the interstitial fluid,
reabsorbed by the lymphatics and subsequently into the blood. In the case of epithelial
cancer types, proteins must break through the basement membrane of the invading tumor
before entering the circulation. Third, as 20-25% of all proteins are secreted, elevated
protein levels may occur due to aberrant secretion or shedding of membrane-bound
proteins containing an extracellular domain (ECD). In addition, single nucleotide
polymorphisms may cause alterations in the signal peptide of proteins resulting in
atypical secretion patterns (80). Cancer-associated glycoproteins may be released into the
circulation due to the change in the polarity of the cancer cells. Also, increased protease
expression may lead to increased ECD cleavage of membrane bound proteins resulting in
increased circulating levels of these cleaved products.

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Proteomics and ovarian cancer

Proteomics focuses on the large-scale determination of gene and cellular function

directly at the protein level. The field of proteomics is a collection of various technical
disciplines, all of which contribute to protein analysis. One powerful proteomic approach
focuses on de novo analysis of proteins or protein populations isolated from cells or
tissues. Studies of cellular proteomes are challenging due to the high degree of
complexity and the low abundance of many proteins requiring highly sensitive analytical
techniques in order to identify these proteins. Among proteomic techniques, mass
spectrometry (MS) has become the main method used in the analysis of complex protein
samples. It has an unparalleled ability to acquire high-content quantitative information
about biological samples of enormous complexity and subsequently to use this data to
identify proteins with high sensitivity and specificity.

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Ovarian cancer proteomics: sources to mine for biomarkers

Potential biomarkers may be identified in various sources such as tumor tissues and
biological fluids such as serum, plasma, disease associated fluid, and cancer cell lines.
These sources may then be analyzed using mass spectrometry in order to identify the
proteins (174). In regards to ovarian cancer, the serum or plasma of ovarian cancer
patients may be compared to the serum or plasma of healthy controls. This biological
fluid is an optimal source to mine for biomarkers, as secreted proteins of the cancer
should be found in the circulation. In addition, if the biomarker is detectable within the
serum of patients and controls, serological tests to measure biomarker levels in plasma
and serum are relatively non-invasive and inexpensive. As the blood contains more than
100,000 different protein forms with abundances spanning over 10-12 orders of
magnitude (4), biomarkers are most likely present in this fluid. Unfortunately, the search
for tumor-derived biomarkers within this fluid is challenging as 20 of the most abundant
plasma proteins (concentration ranges in the mg/mL range) account for 99% of the total
protein mass and impedes the detection of lower abundance tumor antigens by mass
spectrometry (4). Potential tumor markers are expected to exist in the low nanogram to
picogram per millilitre concentration range. However, the presence of highly abundant
proteins such as albumin and immunoglobulins suppresses the ionization of low
abundance proteins. Currently, up-front fractionation techniques are performed in order to
remove major proteins in the blood in order to detect these potential low abundant tumor
markers.

Alternatively, tissue samples from the disease may be another source to mine for potential
biomarkers, such as comparing normal ovarian tissue against ovarian tumors (174).
Hypothetically, certain proteins originating from the tissue could subsequently appear and
be monitored in the blood stream. The shedding and secretion of tumor proteins into the
bloodstream are expected to occur due to leaky capillary beds, protease cleavage and high
rates of cell death within the tumor mass. However, these samples are often complex
incorporating many different types of cells. Often, tumor biopsies may not simply contain
tumor tissues but also include blood components as well as normal tissue. Thus, proteomic
analysis of tumor biopsies may also identify proteins from circulating cells, normal tissues
and from plasma thus only identifying a small population of tumor related proteins (82).

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Hypothesis
Tumors secrete or shed proteins, and these proteins have the potential to enter
circulation. Indeed, the best ovarian cancer biomarkers such as CA-125 and HE4 are shed
and secreted respectively by ovarian tumors and are found in the circulation. It is
reasonable to assume that cell lines derived from ovarian tumors secrete or shed
proteins that are similar to the tumor of origin. Given that conditioned media of ovarian
cancer cell lines are relatively less complex than serum, mining conditioned media
avoids the drawbacks of serum proteomics while providing useful clues to ovarian
cancer biology. We hypothesize that: 1. Proteins secreted or shed by ovarian cancer cell
lines are similar to those secreted or shed by primary ovarian tumours. 2. These proteins
can be identified by two-dimensional liquid chromatography-coupled mass
spectrometry. 3. These proteins can be measured in biological fluids such as serum using
antibody based immunoassays and/or mass spectrometry-based single reaction
monitoring/multiple reaction monitoring assays. 4. Some proteins may serve as
biomarkers for early detection or prognosis of ovarian cancer.


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