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REVIEW

Cancer biomarkers: knowing the present and

predicting the future
Sabarni K Chatterjee &
Bruce R Zetter†
†Author for correspondence
Program in Vascular Biology
Children’s Hospital
300 Longwood Avenue,
Boston, MA 02115, USA
Tel.: +1 617 919 2320
Bruce.Zetter@childrens.harva
rd.edu
In recent years the discovery of cancer biomarkers has become a major focus of cancer
research. The widespread use of prostate-specific antigen in prostate cancer screening has
motivated researchers to identify suitable markers for screening different types of cancer.
Biomarkers are also useful for diagnosis, monitoring disease progression, predicting
disease recurrence and therapeutic treatment efficacy. With the advent of new and
improved genomic and proteomic technologies such as DNA and tissue microarray, two-
dimensional gel eletrophoresis, mass spectrometry and protein assays coupled with
advanced bioinformatic tools, it is possible to develop biomarkers that are able to reliably
and accurately predict outcomes during cancer management and treatment. In years to
come, a serum or urine test for every phase of cancer may drive clinical decision making,
supplementing or replacing currently existing invasive techniques.

Keywords: biomarkers, mass
spectrometry, microarray,
neoplasia, prostate-specific
antigen, proteomics
History of biomarkers
Over the past several decades, considerable
investment has been made in the early detection
of cancer. An increasing number of early cancers
diagnosed as asymptomatic malignancies, or in
some instances even as premalignant lesions, can
be attributed to more efficient screening
programs and changes in clinical practice. The
definitive diagnosis of cancer, has however relied
on histological evaluation of tissues. An ideal
tumor marker would be a protein or protein
fragment that can be easily detected in the
patient’s blood or urine, but not detected in a
healthy person. Today, the most common use of
tumor biomarkers is for detection of early disease
and recurrent disease. In the future, better tests
that may predict tumor outcome in advance and
predict the response of individual tumors to
particular therapeutic drugs may be developed.
The first recognized test for a type of common
cancer was reported in 1965 by Dr Joseph Gold
[1]. He found a substance in the blood of patients
with colon cancer that was normally found in fetal
tissues and named it carcinoembryonic antigen
(CEA). By the end of the 1970s, potential serum
tests had been developed for a variety of cancers
[2]. Additional biomarkers developed in the 1980s
were CA 19-9 for colorectal and pancreatic cancer,
CA 15-3 for breast cancer and CA-125 for ovarian
cancer. However, these early markers have proven
to be reliable indicators of early disease as they are
present in basal levels in normal individuals and
are substantially higher only when there is a con-
siderable amount of cancer present. Furthermore,
these markers are for the most part not specific for
a single cancer. For example, patients with lung or
breast cancer can often have elevated CEA, and
CA-125 can be high in women with
noncancerous gynecological conditions [3].
The best-known cancer biomarker that has
been used by physicians to detect early disease is
the prostate-specific antigen (PSA). The serum
PSA test has been widely used in screening for
prostate cancer in the last decade, and has
brought about a dramatic change increase in early
detection of the disease [4]. The upper limit of
normal PSA level was considered to be 4 ng/ml.
Nevertheless, 33% of tumors spread beyond the
prostate in men, with PSA values between 4 and
10 ng/ml, rendering many of these tumors refrac-
tory to treatment. Between 1989 and 1996 pros-
tate cancer incidence rates increased steadily with
a parallel decrease (2.5% per year) in mortality
rate. The above observations have been attributed
to the dramatic increase in the use of serum PSA
which allowed earlier diagnosis of asymptomatic
prostate cancer [5,6].
Although PSA screening may provide a suspi-
cion of prostate cancer, a clinical diagnosis still
relies on a pathological tissue examination. Of
15 million men screened in 1998 with the PSA
test, 15% or approximately 2.25 million had PSA
levels higher than normal and thus faced the pros-
pect of biopsy [7]. Any healthy individual with a
PSA between 4.0 ng/ml and 10.0 ng /ml is rec-
ommended for biopsy although the lower limit
for suspicion of prostate cancer has been dropped
recently to 2.5 ng/ml. Since PSA elevation is also
associated with benign prostatic hyperplasia
(BPH), elevated PSA levels do not always indicate
the presence of cancer. The resultant specificity of
PSA less than 4.0 ng/ml in detecting early disease

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REVIEW – Chatterjee & Zetter
is just 25%. Consequently, many individuals
undergo an unnecessary biopsy [8].
Despite the advances made in serum PSA
screening, it is still difficult to reliably detect the
early stages of prostate cancer without histologi-
cal examination. Evaluating the Gleason score
from a tissue specimen taken at the time of
biopsy is still the most widely used diagnostic
and prognostic tool for human prostate cancer
[9]. Serum PSA is, perhaps, more reliable as a
marker of prostate cancer recurrence or as an
indicator of treatment efficacy. Levels of PSA in
the blood fall to less than measurable levels (PSA
nadir) after surgical removal of the prostate, rad-
ical prostatectomy, or treatment of prostate can-
cer by radiation [10,11]. This reduction of PSA
blood levels can be monitored over time, and the
finding of a later increase in the level of serum
PSA is considered evidence of a clinical recur-
rence of prostate cancer (PSA recurrence), thus
triggering additional treatment [10]. These values
often predate clinical evidence of prostate cancer
recurrence as determined by radiographic or
physical examination, and are often the only ini-
tial indication of prostate tumor progression [12].
Despite the success of PSA as a prostate cancer
biomarker, useful diagnostic or predictive mark-
ers do not exist for most other tumors. Even in
prostate cancer, the greatest success of PSA is in
detecting recurrent disease. The lack of availabil-
ity of biomarkers with high specificity and sensi-
tivity limits our ability to screen for most cancers.
Sensitivity refers to the percentage of individuals
with disease who are marker positive (validity of a
positive result), whereas specificity refers to the
likelihood that a given marker will be elevated
only in individuals with the disease (validity of a
negative result). A perfect marker for a specific
cancer should have 100% sensitivity and 100%
specificity. PSA for example is a very sensitive
marker but has low specificity. New screening
markers with high specificity and sensitivity are
still required for virtually all cancers.
Use of biomarkers in cancer
The future of cancer management is expected to
be profoundly dependent upon the use of
biomarkers that will guide physicians at every
step of disease management. Cancer biomarkers
can be used for the accurate evaluation and
management of the disease in different stages.
They can be useful for predicting several
outcomes during the course of disease including
early detection, outcome prediction and
detection of disease recurrence. Most
38
importantly, with the clinical appearance of many
new therapeutic agents, appropriate markers can
be used to determine which tumors will respond
to which treatments in order to predict the
likelihood of drug resistance.
Widespread screening
One of the most important roles of cancer
biomarkers will be to utilize them in widespread
screening so that asymptomatic individuals can
be detected with disease at a very early stage. Sur-
prisingly, there are virtually no molecular cancer
markers that are widely recognized as effective
for screening large segments of the population
for the presence of one or more cancers. To date,
the only tumor biomarker approved by US Food
and Drug Administration (FDA) for widespread
screening purposes is PSA along with digital rec-
tal examination (DRE) as discussed earlier.
Despite of the success of PSA in detecting early
stage prostate cancer in some individuals, its use
to screen patients for prostate cancer remains
controversial. Furthermore, it is unclear whether
the benefits of the PSA test outweigh the risk of
follow-up diagnostic tests and treatment. Over-
diagnosis of prostate cancer by PSA screening
can occur where patients with non-life-threaten-
ing small cancers undergo unnecessary complica-
tions resulting from surgery or radiation. Recent
refinements in the PSA test method has made it
possible to distinguish between slow and fast
growing cancers to make the test more specific.
PSA velocity
PSA velocity is the change in PSA level over time.
A steep rise in PSA level increases the likelihood
of malignant prostate cancer. A recent study dem-
onstrated a correlation between the PSA velocity
and time to death from prostate cancer after radi-
cal prostatectomy. Patients whose PSA level
increased by more than 2.0 ng/ml during the year
prior to diagnosis of prostate cancer, were shown
to be at higher risk of dying from the disease
despite undergoing radical prostatectomy [13].
PSA density
PSA density considers the relationship of the
PSA level to the size of the prostate. An elevated
PSA might not arouse suspicion in a patient with
a pre-existing enlarged prostate. Thus,
consideration of prostate density may avoid
unnecessary biopsy in men with elevated PSA
due to benign prostate hypertrophy. The method
has a disadvantage, however, in that some
aggressive cancers may be missed in this cohort.
Future Oncology (2005) 1(1)

Free vs bound PSA
Circulating PSA in the serum has been
identified in two forms, free PSA or PSA bound
to protein. The ratio of free to bound PSA
decreases from benign to cancer i.e., there is
more free PSA in benign conditions whilst more
bound PSA in cancer. Therefore, the ratio of
bound:free PSA can be used as an adjunct to the
total PSA level to provide an additional
indication of the presence of clinically relevant
prostate cancer [14].
Diagnostic & prognostic biomarkers
Although few new markers have reached the
clinic in recent years, technological advances in
genomics and proteomics have produced candi-
date markers that may have potential for cancer
screening. Calcitonin is one of the new tumor
markers that may be used to help early cancer
diagnosis. Calcitonin is elevated in the serum of
a thyroid medullary carcinoma patient and may
with further clinical evaluations, be useful in
screening for this cancer [15]. Calcitonin is a hor-
mone produced by parafollicular C cells in the
thyroid gland that helps to regulate blood cal-
cium levels. Levels of this hormone are elevated
in cancers of the parafollicular C cells, termed
medullary carcinoma of the thyroid. Since med-
ullary carcinoma of the thyroid is often inher-
ited, blood calcitonin can be measured to detect
the cancer in its earliest stages in family mem-
bers who are at risk. Other cancers, particularly
lung cancers, can produce calcitonin, but
measurement of its level in the blood is not
usually used to follow these cancers.
Several reported cancer biomarkers have been
found to have low sensitivity in that they are
found only in a small subset of patients with a
particular type of cancer. Although these
markers are not useful for general screening,
they can be useful in detecting recurrent disease
in those patients whose tumors produce that
particularmarker. One such biomarker is CA-
125, which is present in a subset of ovarian
cancers [16]. CA-125 is also elevated in
endometriosis and some other benign condi-
tions, and fails to identify more than 50% of the
early cancers, thus it is not recommended for
use in a general screening. However, in patients
whose primary tumor is CA-125 positive, post-
surgical elevation of CA-125 levels reflect recur-
rent disease. CEA is a colon cancer marker,
which has low specificity and insufficient
senitivity to be used as a screening marker but is
useful in follow-up.
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Cancer biomarkers – REVIEW
Advances in biomarker discovery
Recent advancements in biomarker
development using gene arrays in addition to
proteomic technologies, including two-
dimensional electrophoresis (2-DE) and mass
spectrometry, have facilitated the discovery of
several new biomarkers. Recently, the FDA has
approved a small number of new urine-based
biomarkers including bladder tumor antigen
(BTA) and nuclear matrix protein-22 as diag-
nostic markers for bladder cancer [17,18]. Celis
and colleagues [19] executed an elaborate pro-
teome analysis using 2-DE and developed a
comprehensive database for bladder cancer. Sur-
vivin, an inhibitor of apoptosis, has also recently
been identified as a urinary diagnostic biomar-
ker for bladder cancer [20]. Another potential
diagnostic biomarker for bladder cancer, calreti-
culin (CRT), has been identified very recently
[21]. These new discoveries provide hope for an
increase in the number of new diagnostic mark-
ers, however the number of biomarkers
approved in the past decade still vastly trails the
number of new therapies that have been
brought to the clinic in that time. Physicians
still depend on invasive techniques including
biopsy or radiologic methods such as mammog-
raphy for early detection of disease. Recent
developments in genomics and proteomics
technologies including mass spectrometry have
provided hope of discovering patterns of multi-
ple biomarkers and/or ‘signature’ protein/gene
profiles specific to each particular cancer [22,23].
In this case, the eventual clinical ‘marker’ may
more accurately be a pattern of genes or pro-
teins that provide an indication of the presence
of cancer in an individual.
The coming decade should see the continued
development of novel biomarkers that will
detect early cancers as well as predict the risk of
early tumors by screening for invasive cancer.
Conceptually, the major difficulty in utilizing
circulating molecular markers as cancer screen-
ing tools is that very small tumors, which need
to be detected and removed prior to metastasis
to other organs, may not produce sufficient
markers for detection in serum or urine. To
make such an early detection possible it is
necessary to first develop new ultrasensitive
methods for detecting very low circulating lev-
els of these analytes. Detecting cancer may also
be compromised if the marker is produced by
any normal tissue and released into the
bloodstream where it would contribute to a
high background signal.
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REVIEW – Chatterjee & Zetter
Predictive biomarkers
In recent years, an increasing number of markers
have been identified that aim to predict cancer
outcome rather than to detect it early. This
progress stems from the ability of new tech-
niques in genomics and proteomics to discover
genes and proteins associated with distinct can-
cer stages. In addition, these markers are often
present in the circulation in detectable amounts,
perhaps due to the larger size of tumors that
require a prognostic test. Such biomarkers can
distinguish between invasive and noninvasive
tumors, between metastatic and nonmetastatic
tumors and between indolent and life-threaten-
ing tumors. Prostate and breast cancer are two
specific cancers that are diagnosed relatively
early compared to other tumors, as a result of
widespread screening programs. Monitoring the
prognosis of these tumors can eventually lead to
a high treatment success rate by providing an
indication as to which patient would benefit
from no treatment, localized treatment such as
surgery or radiation, or early systemic therapy.
Potential prognostic biomarkers have been
proposed for several tumors.
Prostate cancer
As discussed above, PSA has been the major
marker associated with diagnosis of prostate
cancer for the past several years. Despite
improvements to the test, PSA does not provide
a prediction of disease outcome in newly diag-
nosed patients and cannot, by itself, determine
the course of treatment. The great promise of
better prognostic markers is that patients who
are known to be at high risk of future cancer
recurrence, due to the presence of metastatic
markers at the time of diagnosis, could be given
systemic therapy at the time of diagnosis, or
treatment and not wait for the later rise in PSA
which indicative full blown recurrent disease.
Recent interest in the authors’ laboratory and by
other researchers has focused on identifying
biomarkers that distinguish tumors that have
the capacity to metastasize, from those that are
likely to be confined to the primary organ. In
the last decade, the authors’ laboratory has iden-
tified several potential predictive biomarkers
including thymosin β-15, antizyme, antizyme
inhibitor and collagen XXIII that may help to
distinguish metastatic prostate cancer [24–27].
The authors’ early experiments revealed overex-
pression of these genes in metastatic prostate
cancer cell lines. Recently, they tested the
presence of thymosin β-15 in both prostate
40
cancer tissue specimens as well as patient urine,
and established that thymosin β-15 is elevated
in metastatic prostate cancer. In addition, levels
of thymosin β-15 in combination with PSA can
predict recurrence of prostate cancer with more
sensitivity and specificity than PSA alone [95].
Other promising prognostic biomarkers of pros-
tate cancer include the p53 tumor suppressor
gene, bcl-2 proto-oncogene, and Ki-67 prolifer-
ation labeling index. Presence of mutated p53 in
prostate cancer tissue and overexpression of bcl-
2 and Ki-67 are all associated with poor progno-
sis [28,29]. Other potential prognostic markers of
prostate cancer include osteopontin, osteocal-
cin, metalloproteinases (MMPs) and MMP
inhibitors [30,31]. Another recent study has iden-
tified propyl isomerase Pin1 as a potential prog-
nostic marker of prostate cancer. Pin1
expression is directly related to recurrence of
disease. Patients with higher Pin1 expression
have approximately eight times greater probabil-
ity of recurrence than those with low Pin1
expression [32]. Combination of selected prog-
nostic biomarkers, for example thymosin β 15 +
PSA, may ultimately provide the most effective
means for accurately predicting disease
outcome.
Breast cancer
Although mammography is widely used to detect
incipient breast cancer, this technique does not
provide any indication of eventual disease out-
come. Consequently, several recent studies have
sought to identify proteins that contribute to the
‘metastatic signature of breast cancer’ [23]. Two
breast cancer susceptibility genes BRCA1 and
BRCA2 were discovered after research on families
with very strong patterns of breast cancer in 1994
and 1995 [33,34]. These genes are present in all
men and women and are considered to be tumor
suppressors. Women who have a mutation in the
BRCA1 or BRCA2 gene have a significantly ele-
vated risk of developing breast cancer. Genetic
testing for mutations in BRCA1 and BRCA2
genes can be used for predicting the risk of an
individual developing breast cancer. The major
concern regarding the use of these two genes in
accurate prediction of the eventual onset of breast
cancer lies in the variability between individuals.
Identical genetic alterations can lead to a different
disease outcome in different populations of
women, emphasizing modifying factors, such as
diet, smoking and environmental exposures in
determining the final outcome. Furthermore,
women who do not have any of these mutations
Future Oncology (2005) 1(1)

may be given a false sense of security, since
women with no mutation can still be at risk.
Knowing a possible outcome often helps in early
surgical intervention, but it also increases the psy-
chological trauma associated with a prophylactic
surgical intervention. Consequently, BRCA1 and
BRCA2 genetic analysis can best serve as a
prognostic indicator but not as a decision maker.
There are several other factors that can help
monitor prognosis of breast cancer. There is some
evidence that the apoptotic index (the percentage
of dying cells) may be a better predictor of 5-year
disease-free survival than tumor volume, mitotic
index or status of organ confinement. Vascular
density, the marker of blood vessels in a given vol-
ume of tumor, is another important factor that
influences tumor growth and dissemination [35].
Microvessel density (MVD) has been shown to
increase with the declining pathological stage of
the tumor and can predict the likelihood of ext-
racapsular extension especially in breast cancer
[36]. MMPs, which are critical for angiogenesis
and tumor invasion, have also been indicated as
prognostic biomarkers for breast cancer [37].
Absence of estrogen and progesterone receptors,
and upregulation of Ki-67 (Mib-1) antigen are
additional indicators of poor prognosis in breast
cancer patients [38–40]. Another major protein
that can serve as a prognostic marker in several
cancers including breast cancer is osteopontin
(OPN) [41]. OPN is a transformation-associated
protein and has been considered to play a signifi-
cant role as a prognostic biomarker in several can-
cers. High OPN serum levels have been detected
in patients with metastatic cancer of the prostate,
breast and lung [42]. Consequently, patients with
a variety of cancers could benefit from knowing
their level of circulating OPN at the time of
diagnosis as an indicator of the likelihood of
metastatic disease.
Her-2 oncogene expression in tissues or serum
is the most commonly used predictive biomarker
for breast cancer. Expression of Her-2 is signifi-
cantly related to positive lymph nodes, poor
nuclear grade, lack of steroid receptors and high
proliferative activity [43,44]. Her-2 expression in
association with proliferative activity has been
shown to identify a subgroup of node-specific
breast cancer patients with poorer prognosis [45].
Her-2 is a true predictive marker in that its pres-
ence not only provides an indication of disease
outcome, but also can lead to the selection of an
appropriate cancer treatment such as the use of
Herceptin® to treat patients with Her-2 positive
tumors. The combination of Her-2 and
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Cancer biomarkers – REVIEW
estrogen/progesterone receptor status can provide
a greater indication of disease outcome in breast
cancer.
Additional indicators of breast cancer
outcome include urokinase-type plasminogen
activator (uPA) and its inhibitors plasminogen
activator inhibitor (PAI) 1 and -2, [46,47] as well
as levels of lysosomal cysteine proteases cathepsin
B and cathepsin L have also been positively cor-
related to relapse-free survival and overall sur-
vival after treatment of primary breast tumor [48].
The situation with breast cancer highlights the
current status of biomarker application today.
Discovery efforts have revealed several new
biomarkers, however, few are FDA approved and
it is not yet completely understood how these
markers can be used best alone or in combina-
tion to detect or predict outcome in most breast
cancer patients. The next decade should see the
improved application of these and other new
biomarkers to the diagnosis and prognosis of a
variety of tumors.
Ovarian cancer
Ovarian cancer is a target of intense biomarker
research because it is often not discovered until
the disease is quite advanced. CA-125 has been
tested as both a diagnostic and prognostic
marker in ovarian cancer, but it has several lim-
itations including both low specificity and low
selectivity. Barbieri and colleagues have shown
that cyclin D (CD) 1 overexpression is related
to a more aggressive tumor phenotype and poor
prognosis in ovarian carcinoma even though
CD1 expression is not limited to ovarian cancer
[49]. Increased serum concentration of carboxy-
terminal telopeptide of Type I collagen (ICTP)
reflects the aggressiveness of invasive ovarian
cancer [50]. Tumor-associated trypsin inhibitor
is another potentially important predictor of
disease stage in ovarian cancer. Elevated serum
levels and tissue expression in preoperative
patients can be used to predict the stage and
also future prognosis and disease-free survival
[51,52]. Surface-enhanced laser desorption ioni-
zation (SELDI) mass spectrometry technology
has been used to compare serum protein pro-
files of different stages of ovarian cancer [53].
This results in a profile of a large number of
serum peptides that must be sorted and inter-
preted using complex bioinformatic algorithms.
Despite the intense efforts that have been
carried out, to date there is not a reliable
biomarker strategy that allows improved early
detection of this life-threatening disease.
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REVIEW – Chatterjee & Zetter
Colorectal cancer
There have been several studies to identify
genetic markers or signature genetic profiles for
the diagnosis and prognosis of colorectal cancer.
Microsatellite instability that results from
mutations in DNA mismatch-repair genes such
as MLH1, MSH2, or MSH6 is associated with
prognosis of colorectal cancer [54,55]. Increases in
the levels of D-dimer, a fibrin degradation
product, in the serum has also been proposed as
a prognostic biomarker for colorectal carcinoma
[56]. Use of colorectal cancer markers is not
widespread because of their low specificity and
microsatellite instability, which is used as an
indicator for colorectal cancer has minimal
specificity. Rather, it serves as a surrogate
marker for deficiencies in DNA repair that may
be altered in other cancers and disease, rather
than being specific for colorectal cancer [57].
Other cancers
Poor survival rates in non-small cell lung cancer
have also been ascribed to p53 alterations [58].
Other less commonly used prognostic
biomarkers include β-2 microglobulin (β-2M),
which has been used to determine prognosis in
multiple myeloma and lymphomas [59].
Caspase-3 has also been shown to be a novel,
independent prognostic factor in gastric
carcinoma [60]. Recent advances in genomic and
proteomic technologies have identified several
potential marker candidates or protein profiles
from physiological fluids [61].
In spite of these great efforts, there is still a
lack of biomarkers that can accurately predict
the outcome for a specific cancer. Future
emphasis should be placed on identifying
biomarkers that predict the capacity of the
primary tumor to metastasize, the presence or
absence of micrometastatic disease and, where
relevant, the time course of disease recurrence. It
seems unlikely that any single marker will
possess 100% sensitivity and specificity. As new
biomarkers are tested, the use of multiple
independently predictive parameters or
signature protein markers for cancer will
become indispensable in our efforts to improve
cancer management.
Biomarkers for tumor recurrence
Most of the biomarkers in current clinical use
are best applied to demonstrating recurrence. In
many cases, patients who emerge from cancer
surgery and/or chemotherapy will have a 30–70%
statistical chance of a recurrent cancer, due to
42
either local growth or distant metastasis. This
leaves the patient in a state of not knowing
whether they have been cured, or whether they
harbor silent tumors that will grow over time.
Until the promise of prognostic markers is real-
ized, the best choice for these patients is to uti-
lize markers that act as harbingers of recurrent
disease. PSA is a classic example of a biomarker
used for prostate cancer recurrence after initial
nonsurgical treatment. Patients whose PSA lev-
els fall to almost zero after surgery or radiation
can be monitored over time to determine
whether PSA levels remain stable. An increase in
PSA levels at a later time is indicative of
recurrent disease and is generally followed by
systematic therapy.
Additional markers have been discovered that
are associated with the biological behavior of
prostate cancer, helping to move beyond the
reliance on PSA. Interleukin-6 soluble receptor
and transforming growth factor β1 levels are
both measured in patient serum and ‘recurrence
is predicted with the help of a nomogram [62]. In
a separate study it was shown that two biomark-
ers, enhancer of zeste homolog (EZH) 2 and E-
cadherin (ECAD), when found together in
prostate tumor tissue, predicted a threefold
increase in risk of cancer recurrence after
surgery [63].
In other cancers, calcitonin, a biomarker for
thyroid medullary carcinoma is widely accepted
to be a biomarker for thyroid cancer recurrence.
Elevation of calcitonin levels in the serum after
treatment indicates disease recurrence. Thy-
roglobulin is also used to determine cancer
recurrence after the thyroid is removed.
The future of cancer biomarkers
The future of clinical cancer management
belongs to the prognostic and predictive
biomarkers of cancer. These markers are of
utmost importance as they will be the used to
make clinical decisions that will eventually save
lives. In the future, biomarkers will guide deci-
sion making during cancer management.
Biomarker(s) that correctly predict outcome in a
specific disease and allow physicians and
patients to make informed treatment decisions
need to be developed.
The dogma with regard to cancer markers has
been that in the absence of effective treatments,
the best approach to cancer was early diagnosis,
followed by surgical intervention before the
tumor had spread. Although a few effective
techniques such as DRE for prostate cancer,
Future Oncology (2005) 1(1)

needle biopsy and mammography for breast
cancer have been successfully used in the early
diagnosis of cancer, few new diagnostic markers
have emerged. There remains a need for a nonin-
vasive urine or serum marker test that can accu-
rately predict the outcome of cancer. Patients are
still diagnosed too late and treated without
knowing whether their tumor has spread beyond
the primary site.
Over the next few decades, biomarkers will
not only help screen, detect, diagnose, help in
prognostic evaluation, monitor treatment and
predict recurrence, but also play a major role in
clinical decision making. Markers that predict
the response for a given treatment are indeed
needed. The biomarker discovery approach
should include the development of predictors to
determine:
• Treatment or no treatment
• Surgery or no surgery
• Surgery and/or radiation
• Extent of surgical intervention (as in the
choice between mastectomy or lumpectomy
in breast cancer) and most importantly, when
to employ systemic drug therapy
Today, most breast cancer patients in coun-
tries around the world do not receive systemic
therapy until there is evidence of metastatic dis-
ease and this is often too late. In addition,
many new cancer treatments have emerged in
the past decade, but are effective for only a
small fraction of people who have a particular
type of cancer. Thus, the development of these
new treatments has increased the need for
markers that predict outcome and those that
direct which treatment options are most likely
to be effective for a particular patient with a
particular tumor.
New marker development
Concern remains as to whether the tools availa-
ble are well suited to provide the technological
support to meet the demands of new biomarker
development. Until recently, the discovery of
cancer biomarkers has been a slow approach to
identify proteins that are dysregulated as a conse-
quence of the disease and shed into the body flu-
ids such as serum, urine or saliva. Unfortunately,
this approach is arduous and prolonged as each
candidate marker(s) must be identified among
thousands of proteins. The recent advancements
in genomic and proteomic technologies includ-
ing gene array technology, serial analysis of gene
expression (SAGE) improved 2-DE and new
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Cancer biomarkers – REVIEW
mass spectrometric techniques coupled with
advancements in bioinformatic tools, shows
great promise of meeting the demand for the
discovery of a variety of new biomarkers that are
both sensitive and specific.
Genomic approaches
Microarray technology
The use of DNA microarrays has provided one
of the most powerful tools to investigate global
gene expression in all aspects of human cancer.
Microarrays have been used to obtain major
insights into progression, prognosis and response
to therapy on the basis of gene expression pro-
files. Microarray methods were initially devel-
oped to study differential gene expression.
Improvement of the initial methods now permits
more intricate studies that can identify small
deletions or insertions in tumor-suppressor genes
[64], and has been employed to the systematic
analysis of gene expression [65]. One microarray
technology that has drawn widespread use is
GeneChip® (Affymetrix, USA). High-density
oligonucleotide GeneChips are produced by syn-
thesizing several thousands of short oligonucle-
otides in situ on glass wafers, using a
combination of photolithography and light-
directed solid-phase DNA synthesis [66,67]. The
GeneChip series comprises of several different
oligonucleotides to represent each gene on the
array. Each oligonucleotide has a complimentary
sequence with a single base mismatch to account
for nonspecific binding [101]. The obvious advan-
tage of the GeneChip technology is the ability to
measure levels of gene expression in cells and tis-
sues. Its sensitivity allows the detection of very
low abundance mRNA [66]. The major limita-
tion of this microarray technology is that it
requires prior knowledge of the sequence of the
genes to be analyzed. This makes gene prediction
a technical challenge.
SAGE technology is a recent development
that is not only sensitive and comprehensive but
can also analyze gene expression in organisms
with uncharacterized genomes [68]. Several scien-
tists have utilized microarray technology to mon-
itor altered gene expression. Several genes that
were overexpressed following induced expression
of BRCA1 in MDA435 breast cancer cells
included DNA-damage inducible gene
(GADD45) and early growth response 1 (EGR1)
gene. Among the repressed genes were Ki67 and
the prothymosin α gene, which have been
previously been identified as prognostic markers
of breast cancer [69]. Cancer researchers are using
43
REVIEW – Chatterjee & Zetter
SAGE and microarray technology to study
several thousand genes at the same time to
provide insights into diagnosis, prognosis, thera-
peutic targets and clinical outcome [66,70]. Fur-
thermore, there is a significant focus from the
scientific community to identify subsets of dif-
ferentially expressed genes between healthy and
tumor tissues to identify potential biomarker
panels for several cancers including ovarian can-
cer [71], oral cancer [72] and colorectal cancer [73].
Despite serious efforts to minimize variability,
the number of differentially expressed genes that
have been, and will be identified, presents a chal-
lenge in managing and interpreting the data.
Bioinformatic data analysis tools that can inte-
grate microarray data with clinical data are being
developed. This allows investigators to focus on
a smaller subset of genes with direct relevance to
tumor biology. A legitimate concern regarding
mRNA expression study is whether the changes
in mRNA are a true reflection of the expression
of the protein they encode. To date, the most sig-
nificant biomarkers that have emerged from
microarray analysis include estrogen/progester-
one receptor protein expression, HER2
gene/protein alterations, 17q23 genomic ampli-
fications and cyclooxygenase-2 protein expres-
sion, all for breast cancer [74]; insulin-like growth
factor binding protein 2 protein expression for
prostate cancer [75,76]; vimentin protein
expression for kidney cancer [77]; and Myc and
A1B1 protein expression for hepatocellular carci-
noma [78]. However, due to the lack of a unify-
ing bioinformatic resource, most of the data
generated from these studies has remained disor-
derly. Recently, a major collaborative effort has
created ONCOMINE, a cancer microarray data-
base and web-based data-mining platform aimed
at facilitating discovery from genome-wide
expression analyses [79]. To date, ONCOMINE
contains 65 gene expression datasets comprising
nearly 48 million gene expression measurements
form over 4700 microarray experiments. A meta-
analysis study of the database, which includes
approximately 90 datasets from more than 7000
microarray experiments, revealed a signature of
67 genes that appear to be required to change
normal human cells into cancerous ones [80].
Proteomic approaches
Two-dimesional electrophoresis
Previously, two-dimensional polyacrylamide gel
electrophoresis (2D-PAGE) coupled with mass
spectrometry had been the primary proteomic
technology used for biomarker discovery. This is
44
a well-suited technique for direct comparison of
differentially expressed proteins between normal
and tumor tissues and has been used in various
cancers including prostate and breast [81,82].
However, the specificity of the 2D gel technique
remains a concern. Contamination from sur-
rounding tissues present in tissue specimens can
confuse the detection of tumor-specific markers.
Invention of improved tissue-capturing tech-
niques such as laser capture microdissection has
greatly improved the specificity of 2D-PAGE for
biomarker discovery [83,84]. Introduction of tech-
niques that significantly enrich the proteome for
a subset of proteins markedly improves the sensi-
tivity of 2D-PAGE based detection [85]. 2D-
PAGE analyses of normal and malignant tissues
of ovarian cancer identified several overexpressed
proteins including glyoxalase-I and FK506BP
[86]. Recent development of the differential in-gel
electrophoresis facilitates analyses of protein
expression by labeling different populations of
proteins with fluorescent dyes. This technique
has recently been used to identify differentially
expressed proteins in squamous cell carcinoma
and breast cancer [87]. The major limitations of
2D-PAGE methodology are its inability to detect
low abundance proteins and the difficulty of its
application to high-throughput assay.
Mass spectrometry
Mass spectrometry has been used traditionally
with 2D-PAGE as a means to identify the spots
that appear on the gel. Recent technological
developments make mass spectrometry an impor-
tant tool on its own for the rapid identification of
cancer biomarkers. Mass spectrometry analysis of
a complex proteome is sensitive, robust and
quantitative. Matrix-assisted laser desorption ion-
ization time-of-flight (MALDI-TOF) mass spec-
trometry and tandem mass spectrometry are
routinely used in the identification of biomarkers
coupled with 2D electrophoresis. Recent techno-
logical improvements in mass spectrometry
instrumentation have increased their use in
biomarker discovery immensely. Direct analysis
of biological samples using mass spectrometry is
becoming more popular due to its high-through-
put nature and increased sensitivity. The authors’
laboratory have recently identified over 400 pro-
teins differentially regulated in metastatic pros-
tate cancer cells by the quantitative method of
stable isotope labeling with amino acids in cell
culture (SILAC) mass spectrometry [88]. Using
SILAC, cells representing two different biological
conditions are cultured in amino-acid-deficient
Future Oncology (2005) 1(1)

growth media supplemented with 12C (light) or
13C (heavy) labelled amino acids and proteins.
Fractions from each culture are then mixed and
subjected to traditional tandem mass spectrome-
try. This method quantifies the difference in
expression of individual proteins in the two dif-
ferent conditions by calculating the ratio of the
intensities of corresponding peaks containing
heavy and light amino acid. Using another mass
spectrometry based technique, isotope-coded
affinity tag (ICAT) clinically distinct samples can
be quantitatively analyzed by chemically labeling
the proteins with heavy or light tags that bind to
cysteine residues in the proteins. This technology
has been used to identify and quantify expression
of proteins from cells, tissues or other biological
fluids [89]. The ICAT technique has been success-
fully used for quantitative studies of markers
associated with androgen stimulated and
unstimulated cancer cells [90].
Another addition to the mass spectrometry
technology is the surface-enhanced laser
desorption ionization time-of-flight (SELDI-
TOF) mass spectrometry. This technology has
the potential to very rapidly compare biomarker
patterns in tissues and body fluids. Proteomic
profiles of body fluids such as serum or urine of
cancer patients can be analyzed in a time efficient
manner by the use of SELDI-TOF mass spec-
trometry. This technology has been effectively
used in the validation of serum prostate-specific
membrane antigen as one of the markers that dis-
tinguishes prostate cancer from benign disease
[91]. A proteomic pattern that distinguished dif-
ferent stages of ovarian cancer was identified by
this technology [92]. Its wide acceptance is still in
question due to some concerns regarding repro-
ducibility and reduced sensitivity at high molecu-
lar weight range [93]. Table 1 summarizes the
important biomarkers and their use as discussed
in this article.
Sources for marker testing
A very important aspect of marker development
is to translate its usefulness to the clinic. A poten-
tial marker can be tested in different sources,
including tumor tissues and body fluids such as
serum and urine. The methodologies should be
of rapid execution, reliable and possibly not very
expensive. Several techniques that are used in
biomarker assays will be discussed.
Immunohistochemistry
Immunohistochemistry is a technique that
utilizes the staining of histological specimens for
www.futuremedicine.com
Cancer biomarkers – REVIEW
a particular marker. Immunohistochemistry is
widely used to predict outcomes among patients
with different grades of disease by staining tis-
sues from biopsy samples taken from cancer
patients at the time of diagnosis. Major disad-
vantages of this method are that it depends on
biopsy samples that are collected using invasive
techniques, and the small percentage of tumor
tissue obtained during biopsy sometimes misses
more aggressive tumor populations that reside
nearby.
Enzyme-linked immunosorbent assay
A marker is applicable as a fluid analyte when at
least two requirements are met: the potential
marker must circulate in the serum or urine and
a quantitative high-throughput assay must be
available to detect the marker. Enzyme-linked
immunosorbent assay (ELISA) is a sensitive,
high-throughput technique that quantitatively
measures the amount of analyte present in a
physiological fluid such as serum or urine. The
advantage of ELISA is that it can be utilized with
body fluids that are collected noninvasively. In
brief, the primary antibody binds to the analyte,
and an enzyme-linked secondary antibody binds
to the previous complex. The enzyme activity is
quantitatively measured by the addition of an
appropriate substrate, and it is proportional to
the amount of analyte present in the fluid. Varia-
tions include the competitive ELISA and the
sandwich ELISA. This technique is widely
accepted as a clinical tool and is very sensitive.
Chip technology
With the emergence of genomics and proteomics
research in the last few years, it has become
necessary to develop techniques that can provide
comparative information between several differ-
ent models simultaneously. With the discovery
of several markers in recent years and the grow-
ing belief that a panel of markers rather than one
marker alone will predict a more accurate out-
come, development of new detection systems
have become necessary. There is a need for tools
that can do for protein expression profiling what
DNA chips have done for a RNA expression
analysis. Analyzing protein expression can aid
researchers in understanding the molecular basis
of disease, including disease susceptibility,
diagnosis, progression and potential points of
therapeutic interference. This has led to a surge
in the development of ‘protein chips’. The basic
format of most protein chips is similar to DNA
chips, such as use of a glass or plastic printed
45
REVIEW – Chatterjee & Zetter

containing a panel of molecules such as

Table 1. Cancer biomarkers and their use.
antibodies would be able to predict a cancer state
Biomarkers Cancers Use Refs by a simple serum or urine test. This technology
PSA Prostate Screening, diagnostic, [4] is likely to see considerable application and
predict recurrence development in coming years.
CEA Several cancers Determine recurrence, [1]
including Monitor treatment Limitations of biomarker development
colorectal, lung, efficacy The critical limitation in biomarker development
breast, liver, is the lack of a proper structure in the biomarker
pancreatic, discovery process as is present in testing a new
thyroid,bladder
drug. Recently, the establishment of the Early
CA 125 Ovarian Diagnostic, monitor [16]
Detection Research Network (EDRN) by the
treatment, predict National Cancer Institute, USA has led to the
recurrence
improved coordination between biomarker
BTA Bladder Diagnosis, predict [17]
research laboratories. Pepe and colleagues pro-
recurrence.
posed a five-phase formal categorization that
NMP22 Bladder Diagnosis, predict [18]
would guide the development of a biomarker [94].
recurrence The structure of the biomarker development
Calreticulin Bladder Diagnosis [21] process has been outlined in Table 2.
Survivin Bladder Diagnosis [20] Most of the biomarkers currently known are of
Calcitonin Thyroid Diagnosis, monitor [24] limited clinical use. Early studies lacked epidemi-
treatment and predict ological validity or statistical power and thus
recurrence lacked universal application to populations. Lack
Antizyme Prostate Prognosis [25] of pre-analytical studies and standardized proto-
Antizyme inhibitor Prostate Prognosis [26] cols across laboratories adds to diminished repro-
Collagen XXIII Prostate, breast Prognosis [27] ducibility. These problems render many markers
several others insufficiently sensitive or specific enough for clin-
MMP Prostate, breast Prognosis [41,42] ical use. The application of uniform standards as
suggested by Pepe should facilitate the translation
MMP inhibitors Prostate, breast Prognosis [30]
of newly discovered biomarkers to the clinic [94].
Her-2 Breast Prognosis, response to [31]
therapy
Future perspective
Urokinase-type Breast Recurrence [47]
As more potential biomarkers are discovered, the
plasminogen
limitations in clinical use of these new markers,
activator
in the discovery phase, are reduced and more in
PAI-1, PAI-2 Recurrence [47,48]
the validation of the markers and rapid applica-
Cathepsin B and L Breast Recurrence [50]
tion to clinical practice. Oncology practice in the
Cyclin D1 Ovarian Prognosis, recurrence [51] next decade will be ruled by cost effectiveness.
ICTP Ovarian Prognosis, stage [60] Biomarkers that detect cancers, predict cancer
β-2 microglobulin Multiple myeloma Prognosis [61] outcome and influence treatment choice will
and lymphoma have a major role in determining cost effective-
Caspase-3 Gastric carcinoma Prognostic ness in clinical cancer management. Biomarkers
EZH2 Prostate Recurrence [64] will be of greatest importance if they can focus
Vimentin Kidney Prognosis [78] the use of expensive cancer treatments on those
who are most likely to benefit. This requires the
Myc and A1B1 Hepatocellular Prognosis [79]
carcinoma
methods to be simple, inexpensive, robust and
reliable. To determine disease outcome, no single
SELDI pattern Ovarian cancer Diagnosis, prognosis, [93]
biomarker is likely to have the appropriate degree
stage
of certainty to dictate treatment decisions. Con-
BTA: Bladder tumor antigen; CEA: Carcinoembryonic antigen; EZH: Enhancer of zeste
homolog; ICTP: Carboxy terminal telopeptide of type I collagen; MMP: Matrix
sequently, the future of cancer prognosis may rely
metalloproteinases; PAI: Plasminogen acitvator inihibitor; PSA: Prostate-specific on small panels of 6–10 markers that can give an
antigen; SELDI: Surface-enhanced laser desorption ionization. accurate molecular staging that will indicate the
likelihood of metastatic involvement and the
with an array of molecules, such as antibodies need for rapid systemic therapy. The high-
that can capture proteins. Ideally, a protein chip throughput technology platforms will help in the

46 Future Oncology (2005) 1(1)

 Cancer biomarkers – REVIEW

microarray-chip technology as well as the 2D gel

Table 2. Phases of biomarker development.
Preclinical exploratory Phase 1 Promising directions identified
Clinical assay and validation Phase 2 Clinical assay detects established
disease
Retrospective longitude Phase 3 Biomarker detects disease early
before it becomes clinical and a
"screen positive" rule is defined
Prospective screening Phase 4 Extent and characteristics of
disease detected by the test and
the false referral rate are identified
Cancer control Phase 5 Impact of screening on reducing
the burden of disease on the
population is quantified
discovery of multiple new biomarkers for a
particular disease simultaneously. At present, the
and mass-spectrometry, are not easily applicable
to the clinical setting and require well equipped
laboratories and well-trained personnel. Simple,
rapid and sensitive microarray-based protein
chips, label-free detection systems and antibody-
based protein chip systems that will bring the
advancements of biomarker discovery into clini-
cal practice are in development. We are
approaching a time when the use of proper
biomarkers will help detect cancer, monitor and
manage progression of the disease and its thera-
peutic treatment. Development of simple diag-
nostic kits that will accurately and reliably
predict cancer and can be used in the clinic or, by
potential patients themselves is a crucial goal for
the future of oncology.

Executive summary
Biomarker history
• Biomarkers developed in the 1980's were CA 19-9 for colorectal and pancreatic cancer, CA 15-3 for breast cancer and CA-125 for
ovarian cancer.
• Prostate-specific antigen (PSA) is the best-known cancer biomarker that has been used by physicians to detect early disease.
• Serum PSA is, perhaps, more reliable as a marker of prostate cancer recurrence or as an indicator of treatment efficacy.
• Levels of PSA in the blood drop to less than measurable levels (PSA nadir) after surgical removal of the prostate (radical prostatectomy) or
treatment of prostate cancer by radiation.
• Even in prostate cancer the greatest success of PSA is in detecting recurrent disease.
Use of biomarkers in cancer
• Most important role of cancer biomarkers is to utilize them in widespread screening so that asymptomatic individuals can be detected
with disease at a very early stage.
• Prostate-specific antigen (PSA) along with digital rectal examination (DRE) is the only FDA approved screening biomarker.
• PSA velocity, PSA density and Free versus bound PSA are better predictors than PSA alone.
• Calcitonin, a thyroid cancer marker can be a potential screening biomarker.
• Recently, the FDA has approved a few diagnostic bladder cancer biomarkers such as NMP 22 and BTA.
• Advancements in genomics and proteomics technologies facilitated the discovery new potential markers.
• Genetic and proteomic signature profiles have been used as potential predictors of disease outcome.
Future of biomarkers
• The future of clinical cancer management belongs to the prognostic and predictive biomarkers of cancer.
• Development of new treatments has increased the need for markers that predict outcome and those that direct which treatment options
are most likely to be effective for a particular patient with a particular tumor.
New marker development
• The recent advancements in genomic and proteomic technologies including gene array technology, improved two-dimensional gel
electrophoresis and new mass spectrometric techniques coupled with advancements in bioinformatic tools shows great promise of
meeting the demand for the discovery of a variety of new biomarkers that are both sensitive and specific.
Sources of biomarker discovery
• Several techniques that are used in biomarker assays include immunohistochemistry (IHC), enzyme-linked immunosorbent assay (ELISA)
and Protein Chip Technology.
Limitations of biomarker development
• The critical limitation in biomarker development is the lack of a proper structure in biomarker discovery process as is present in testing a
new drug.
• The establishment of Early Detection Research Network (EDRN) by the National Cancer Institute of USA has led to the improved
coordination between biomarker research laboratories.
• Phases of biomarker discovery have been outlined by the EDRN.
Future perspective
• Biomarkers that detect cancers, predict cancer outcome and influence treatment choice will have a major role in determining the cost-
effectiveness in clinical cancer management.
• A small panel of biomarkers collectively will predict accurate molecular staging of disease.
• Development of simple diagnostic kits that will accurately and reliably predict cancer and can be used in the clinic or, by potential patients
themselves is a crucial goal for the future of oncology.

www.futuremedicine.com 47
REVIEW – Chatterjee & Zetter
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50
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Website
101. www.affimetrix.com
(Accessed January 2005)
Affiliations
• Bruce R Zetter
Children’s Hospital, 300 Longwood Avenue,
Boston, MA 02115, USA
Tel.: +1 617 919 2320
Bruce.zetter@childrens.harvard.edu
• Sabarni K Chatterjee
Program in Vascular Biology, Children’s Hospital,
Boston and Harvard Medical School, Boston,
MA 02115, USA
Sabarni.Chatterjee@childrens.harvard.edu
Future Oncology (2005) 1(1)