834 The Immunoassay Handbook
fecal occult blood/hemoglobin for colorectal cancer, CA
125 for ovarian cancer, prostate-speciﬁc antigen (PSA) for
prostate cancer, and p21 ras oncoprotein. Prostate cancer
screening using PSA and digital rectal examination was
recommended by the American Cancer Society in Novem-
ber 1992 for men over 50 years of age. However, it remains
controversial, and in spite of this strong endorsement, a
signiﬁcant number of oncologists question the merits of
screening as related to treatment options and survival ben-
eﬁt. In 2012, the US Preventive Services Task Force
(USPSTF) recommended against PSA-based screening
for prostate cancer. Another prominent marker worth
mentioning is the identiﬁcation of mutated forms of
hereditary cancer susceptibility BRCA1 and BRCA2 genes
or gene products, which identiﬁes high-risk individuals.
In 2003, a new two-in-one test was approved by the
FDA for the primary screening of cervical cancer. It
includes a sandwich immunoassay test with the traditional
50-year-old Pap smear for women over the age of 30. The
Pap test is widely used as a screening test that is done
annually to detect the presence of abnormal cellular mark-
ers indicative of cancerous or precancerous conditions.
This was developed by George Papanicolaou and involves
the collection of vaginal ﬂuid or cells scraped from the cer-
vix to predict or detect cervical cancer. The new compo-
nent of the combined test (Digene, QIAGEN, USA) is the
genetic test for 13 strains of the human papilloma viruses
(HPVs), which are largely sexually transmitted and the
likely cause of 99% of cervical cancers. While millions of
women are infected with HPV, only those above the age of
30 and having persistent infections are at high risk. The
Hybrid Capture® 2 DNA test uses a two-RNA probe mix-
ture to distinguish between the carcinogenic and low-risk
HPV types. The company offers a speciﬁc specimen col-
lection device to accompany the combined test procedure,
although it is similar to the traditional sample collection
method. The principle of the hybrid capture method is
essentially a sandwich immunoassay that detects the spe-
ciﬁc DNA–RNA hybrid as the antigen. The specimen is
dissolved in a base solution to dissociate the various com-
ponents including the target viral DNA. The speciﬁc HPV
RNA probe is added to form a DNA–RNA hybrid, which
is selectively captured by a solid phase coated with an anti-
body with speciﬁcity to the hybrid nucleic acids. The cap-
tured DNA–RNA hybrid is detected by an alkaline
phosphatase (ALP)-labeled antibody and detected by che-
miluminescent dioxetane substrate. The combination test
has been found to have better sensitivity than the use of
either test alone. In some countries outside the USA, the
HPV test is also used as a primary screening test alone or
in conjunction with the Pap test.
Almost every cancer marker has been investigated for its
suitability as a primary diagnostic test for cancer in symp-
tomatic individuals. However, sufﬁcient false positives
and false negatives have been encountered with every
marker so far discovered to preclude their use in distin-
guishing malignant and nonmalignant conditions. The
ultimate goal of identifying tumor-speciﬁc antigens has so
far eluded oncologists because most tumor markers have
been found in some normal tissues and the serum of some
noncancerous individuals and in many benign diseases.
For this reason, these antigens are often referred to as
tumor-associated antigens. Nevertheless, a number of
cancer markers have proved to be useful in conﬁrming
diagnosis, often in conjunction with a battery of other
clinical methods. Another approach attempts to use mul-
tiple tumor markers to diagnose tumors and to identify
the primary origin of metastic disease (Wu and Naka-
mura, 1997; Hanausek and Walaszek, 1998). This is in
parallel with the more frequent use of drug combinations
to treat tumors.
Immunoassays for some cancer markers are used in clinics
to distinguish between clinical conditions with similar
symptoms, where one or both could be cancerous. For
example, the measurement of neuron-speciﬁc enolase
(NSE) levels allows differentiation between neuroblas-
toma and Wilm’s tumor when a child presents with a pal-
pable abdominal mass. Similarly, PSA and prostatic acid
phosphatase (PAP) can distinguish prostate cancer metas-
tasis from other secondary tumors whose primary origin is
not the prostate gland. Antibody probes speciﬁc for B or T
cells can establish the lineage and classify leukemias and
lymphomas in an immunohistochemical assay (see HEMA-
TOLOGY). Antibodies speciﬁc to lymphoid malignancies
can distinguish between non-Hodgkin’s lymphoma and
undifferentiated cancer of nonhematopoietic origin.
IN CANCER DIAGNOSIS
Highly sensitive diagnosis and accurate analysis of bio-
markers in human samples are important for the early
detection, treatment, and management of cancer. For a
traditional immunometric (sandwich) immunoassay that
is routinely used for protein biomarker identiﬁcation, a
capture antibody against a speciﬁc biomarker is ﬁrst
immobilized on a 96-well plate. After the binding of anti-
gen, a labeled detection antibody is allowed to bind with
the immobilized antigen. The concentration of the anti-
gen can then be determined by indirectly measuring the
concentration of the probe attached to the detector anti-
body, which may include enzymes, ﬂuorescence tags,
DNA barcodes, etc. A heterogeneous immunoassay
involves antibody immobilization, multiple steps of incu-
bation and washing cycles, followed by signal ampliﬁca-
tion and reading. From the initial antibody immobilization
to the ﬁnal reading of the assay results, the immunoassay
may take from hours to days to complete. A traditional
immunoassay is rather time and labor intensive. To over-
come these problems, the development of single-step,
washing-free homogeneous immunoassays has gener-
ated a lot of interest and value to the scientiﬁc commu-
nity. The magnitude of light scattering by a gold
nanoparticle can be signiﬁcantly higher than light emis-
sion from strongly ﬂuorescing dyes. This unique charac-
teristic has led to many promising applications of metal
nanoparticles in the biomedical ﬁeld including molecular
835CHAPTER 9.13 Cancer Markers
and cell imaging, biosensing, bioassays, and photothermal
therapy. By taking advantage of the large scattering cross-
section of gold nanoparticles and the high sensitivity of
“dynamic light scattering measurement,” biomarker pro-
teins or other biomolecular targets can be detected at very
low concentrations using gold nanoparticle probes. Uti-
lizing this methodology, biomarker proteins were detected
by Liu et al. (2008) at very low concentration using gold
The development of thyroglobulin (Tg) assays and
recombinant human TSH (rhTSH) has provided the
means for physicians to identify residual (or recurrent)
thyroid cancer much earlier than few decades ago, when
diagnostic technologies were limited to physical examina-
tion, 131I scans after T4 withdrawal, and chest X-rays. The
combined use of serum Tg and whole-body scan after
rhTSH stimulation is particularly effective in the detec-
tion of small residual foci of thyroid tissue. A general
assumption is that earlier detection and treatment of recur-
rent disease should lead to better outcomes, but proper
evidence is absent in that 131I therapy of microscopic dis-
ease helps the low-risk patient whose survival approaches
100%. Smallridge et al. (2007) reported that using a Tg-
immunoassay with a cutoff 5- to 10-fold lower than
reported by others, it is possible to follow patients with
differentiated thyroid cancer and a T4-suppressed Tg
below 0.1ng/mL without the need to perform rhTSH
stimulation, which also has signiﬁcant economic beneﬁts.
Gong et al. (2007) developed a simple, sensitive, and
speciﬁc immunoassay, based on surface-enhanced Raman
scattering for human AFP, a tumor marker for the diagno-
sis of hepatocellular carcinoma. This methodology com-
bined Ag/SiO2 core–shell nanoparticles with rhodamine B
isothiocyanate dye molecules as Raman tags and used
amino group-modiﬁed silica-coated magnetic nanoparti-
cles as the solid-phase immobilization matrix and separa-
tion tool. The system involved an immunometric
(sandwich)-type immunoassay between polyclonal anti-
body-functionalized Ag/SiO2 nanoparticle-based Raman
tags and monoclonal antibody-modiﬁed silica-coated
magnetic nanoparticles. The presence of the analyte and
the antibody–antigen reaction can be monitored by the
Raman spectra of the Ag/SiO2 tags. The advantages of this
novel strategy include the high stability of Raman tags
obtained from the silica shell-coated silver core–shell
nanostructure and the use of silica-coated magnetic
nanoparticles as immobilization matrix and separation
tool, avoiding pretreatment and washing steps. This assay
is able to measure human AFP concentrations up to
0.12µg/mL with a detection limit of 11.5pg/mL.
A rapid and reproducible surface-enhanced Raman scat-
tering (SERS)-based immunoassay technique, using hol-
low gold nanospheres (HGNs) and magnetic beads, has
also been developed by Chon et al. (2009) for the detection
of lung cancer marker carcinoembryonic antigen (CEA).
Gold et al. (2006) developed a new MUC1 serum immuno-
assay that is able to differentiate cancer from pancreatitis.
An enzyme immunoassay was established with mAB PAM4
as the capture antibody and a polyclonal anti-MUC1 anti-
body as the detection probe. Patient sera were obtained
from healthy, adult patients with acute and chronic pan-
creatitis, and those with pancreatic and other forms of
cancer, and were measured for PAM4-reactive MUC1.
The sensitivity and speciﬁcity observed suggest that the
PAM4-based immunoassay of circulating MUC1 may be
useful in the diagnosis of pancreatic cancer.
STAGING AND GRADING
The degree of elevation in the concentration of several
tumor markers can help to stage tumors. In general, the
mean circulating levels of these tumor markers increase
with the stage of the cancer. In contrast, placental ALP
(PLAP) is a tumor marker related to the grade of cancer,
and serum levels of this analyte are higher in Grade 1 and
2 tumors than in Grade 3 ovarian carcinomas.
Prognosis is the probability of cure of a cancer patient.
Positive lymph node detection is a classical method of
determining prognosis invasively. The magnitude of tumor
marker levels in several cancers corresponds to the mass of
tumor. Moderate elevations are suggestive of better prog-
nosis than persistent high levels. An important prognostic
factor in ovarian and breast cancers is the ampliﬁcation of
the c-erbB-2 gene (HER-2/neu) and protein. Tumor
aggressiveness resulting in widespread metastasis precipi-
tates very high serum tumor marker levels, indicating poor
prognosis. Generally, well-differentiated tumors tend to be
less aggressive than undifferentiated or anaplastic tumors.
While most tumor marker overexpressions indicate poor
prognosis, the increased levels of progesterone and estro-
gen receptors in breast cancers determine the type of treat-
ment (hormone) as well as good prognosis.
MONITORING AND RECURRENCE
The proﬁle of tumor marker concentration against time
can mirror the condition of patients diagnosed to have
cancer, for example indicating whether therapy has been
successful or if remission has occurred (see Fig. 1). This is
one area where tumor markers are most useful (Pannall
and Kotasek, 1997; Suresh, 1996).
Tumor marker proﬁles usually reﬂect one of the follow-
ing classical patterns:
G A rapid decline in tumor marker level to normal concen-
trations following surgery or other forms of ﬁrst-line
therapy suggests that treatment has been successful.
G The lack of a decline to basal levels following ﬁrst-line
therapy may indicate that treatment has only been par-
G Continued low levels of the tumor marker indicate that
remission has been maintained as a result of treatment.
G A subsequent rise in the concentration of the tumor
marker (from the basal level) suggests a recurrence of
the disease. Tumor markers can warn of renewed
tumor growth or recurrence 3–12 months before other
methods provide conﬁrmation.
G Decline of the marker levels after an increase has been
associated with a recurrence, suggestive of the respon-
siveness of a tumor to second line or subsequent
836 The Immunoassay Handbook
G If tumor marker concentrations remain elevated after
treatment, the tumor may be resistant to the therapeutic
method employed and the prognosis of the patient is poor
unless alternative therapeutic modalities are available.
These characteristic proﬁles can be observed for many
tumor markers, e.g., CEA in colorectal cancers, cancer
antigen 125 (CA 125) in ovarian cancers, or PSA in pros-
Although these classical patterns in tumor marker pro-
ﬁles are seen in the majority of patients, they do not reﬂect
the clinical status of every patient. Hence, some oncolo-
gists recommend the estimation of more than one marker
(Wu and Nakamura, 1997). For example, in pancreatic
cancer, carbohydrate antigens 19-9 (CA 19-9), 50 (CA 50),
and CEA may all be elevated. However, CA 19-9 is posi-
tive in 75% of the patients, whereas CEA is positive in less
than 50%. In certain germ cell tumors, the combined mea-
surement of human chorionic gonadotropin (hCG) and
AFP is desirable to conﬁrm diagnosis and manage patients.
The association of multiple tumor markers with a range of
cancers is shown in Fig. 2.
FIGURE 2 The association of tumor markers with different cancer sites.
FIGURE 1 Classical trends in tumor marker proﬁles: (a) successful ﬁrst-line therapy with reduction to normal levels; (b) unsuccessful ﬁrst-line
therapy or partial response; (c) continued clinical remission; (d) recurrence of cancer; (e) response to second line of therapy; (f) failure of or resistance
to therapy with poor prognosis.
837CHAPTER 9.13 Cancer Markers
With the advent of automated multi-analyte analyzers
for tumor markers, the assay of more than one marker is
desirable and straightforward to carry out. This provides
greater conﬁdence in establishing clinical status and may
become a feature of the use of tumor markers in oncology
in the future.
A welcome adjunct is the availability of tumor marker
reference controls for several analytes like CA 19-9, CA
125, CA 15-3, and CEA from Bioref in Germany and Bio-
Rad in North America. Randox has a liquid tumor marker
control containing 15 cancer-related markers providing a
comprehensive coverage of well-known markers.
History and Classification
The history of the discovery of biochemical tumor mark-
ers starts with the description of a urinary substance in
1846, which is now known to be excessive secretion of the
immunoglobin light chain in multiple myeloma. The next
hundred years saw sporadic description of hormones
(hCG, ACTH), enzymes and isoenzymes (AP, PLAP), and
cytokeratins (TPA) as potential tumor markers. The devel-
opment of key techniques in the latter half of the twentieth
century was important for the rapid discovery and devel-
opment of new tumor markers and their immunoassays.
Central to the theme of this entire book is the develop-
ment of the immunoassay concept in the 1950s by Yalow
and Berson, involving the application of antibodies as
reagents to measure speciﬁc substances in complex mix-
tures. Numerous immunoassays emerged using polyclonal
antibodies subsequent to this period, although most were
to measure noncancerous analytes. It was in the early
1970s that the CEA immunoassay was introduced as a
commercial test for cancer. MAb techniques introduced in
1975, and the development of the immunometric (sand-
wich) immunoassay format in 1982, revolutionized the
ﬁeld of tumor markers. This resulted in a virtual explosion
in the discovery of new tumor antigens and the introduc-
tion of several among them as immunoassays for routine
clinical use. Recombinant antibody techniques also pro-
vided invaluable insights into the understanding of the
structure and putative functions of tumor markers. Molec-
ular biology techniques were key to the recent discovery of
several emerging tumor markers belonging to the classes
of oncogenes, tumor suppresser genes, and a host of other
molecules including angiogenic factors, cyclins, nuclear
matrix proteins (NMPs), cell adhesion factors, heat shock
proteins, growth factors and their receptors, and telomer-
ase (Wu and Nakamura, 1997; Suresh, 1996).
Almost every new major, and many minor, molecule dis-
covered in the last 10 years has been investigated as a
potential new tumor marker. Hence, it is beyond the scope
of this chapter to describe all of these new tumor markers,
and the reader is directed to an excellent book devoted to
this topic (Wu and Nakamura, 1997).
A classiﬁcation of tumor markers is provided in Fig. 3.
Almost all tumor markers are now considered tumor-
associated antigens, due to their expression to some extent in
some noncancerous tissues. However, rare examples of
highly speciﬁc tumor antigens can be recognized as a
separate subgroup. These include the B-cell tumor
immonoglobulin idiotype (the unique paratope or variable
region of the speciﬁc immunoglobulin expressed on the sur-
face or as a secreted myeloma protein), T-cell receptor of
T-cell leukemia, mutated forms of oncogenes and tumor
supressor genes, and several virus-induced antigens found
predominantly in nonhuman cancers. Tumor-associated
markers can be classiﬁed into two subcategories based on
the size of the molecules. The macromolecular markers
have several categories including proteins, genes, chromo-
somes, and histologically identiﬁable cellular markers.
NOMENCLATURE AND IDIOSYNCRASIES
OF GLYCOPROTEIN TUMOR ANTIGENS
The discovery of several new large glycoprotein tumor
markers has been fueled by mAB technology. However, a
word of caution is relevant at this juncture to illustrate the
misuse of mAb technology. Claims of discovery of a new
tumor marker are abundant, based mainly on the develop-
ment of a new monoclonal and its putative recognition of a
new tumor antigen. Several monoclonals have been devel-
oped measuring complex and large glycoproteins elevated in
breast, ovarian, pancreatic, gastric, lung, and colorectal
cancers. The nomenclature of many of these cancer antigens
(CAs) has been derived from the arbitrary clonal designa-
tions of the various mAbs. For example, the mAb OC125
was developed by immunizing mice with human ovarian
cancer cells (Bast et al., 1981). The antigen identiﬁed by this
mAb is now recognized as CA 125. Similarly, CA 19-9
antigen was originally identiﬁed by the NS19-9 mAb. Some
confusion has been introduced in the tumor marker
FIGURE 3 Classiﬁcation of tumor markers.
838 The Immunoassay Handbook
literature, due to the development of several different
mAbs to overlapping or distinct epitopes on the large gly-
coprotein antigens with claims of identifying new markers,
with increased clinical sensitivity/speciﬁcity of one over
the other. Adding to this confusion is the question of what
is measured by these glycoprotein tumor marker assays—
epitope, antigenic determinant, domain, or the antigen. It
is sufﬁcient to state that every tumor marker assay mea-
sures antigens, albeit via unique mAbs binding to unique
epitopes. New tumor marker antigens/epitopes have been
described purely based on the development of a new mAb
without making serious attempts to compare and contrast
it with preexisting mAbs and antigens. This important
issue is not easy to resolve due to the idiosyncrasies of the
large cancer glycoproteins. Unlike traditional analytes,
these large cancer glycoproteins and mucins have special
features (Suresh, 1991). The precise estimation of these
glycoproteins is inﬂuenced by pH, valency, and distribu-
tion or density of the epitope and serum anti-carbohydrate
antibodies. An anomalous feature unique to these analytes
is that many cancer serum samples exhibit increased recov-
ery/estimation of the antigen upon dilution. It is not often
appreciated that human serum has a substantive amount of
anti-carbohydrate IgM and lgG antibodies that can inter-
fere with glycoprotein immunoassays. It appears that
in serum, these large cancer glycoproteins can exist as
supramacromolecular complexes promoted partly due to
glycan–glycan interactions and partly as a result of weak
cross-linking by anti-carbohydrate antibodies. This
hypothesis (Suresh, 1991) explains why upon dilution or
lowering the pH of serum during assay (e.g., CA 19-9 kit,
Fujirebio—previously Centocor), one can recover higher
amounts of the antigen in an immunoassay, presumably
due to disassociation of the large complexes. This concept
also can explain the often observed phenomenon of co-
expressions of CA 125, CA 19-9, CA 15-3, sialyl LewisX,
LewisX, LewisY and other antigens.
An attempt to critically study some of these complex
issues has been initiated by the International Society For
Oncodevelopmental Biology and Medicine (ISOBM).
They initiated tissue differentiation (TD) workshops a few
years ago, analogous to the CD workshops to classify leu-
kocyte antigens. These workshops have been conducted
for several tumor markers such as CEA (Hammarstrom
et al., 1989; Nap et al., 1992), CA 125 (Nustad et al., 1996;
Nap et al., 1996), AFP (Alpert and Abelev, 1998), PSA
(Stenman et al., 1999), MUC1 (Price et al., 1998), cyto-
keratins (Stigbrand et al., 1998), and sialyl Lewis A (Rye
et al., 1998), primarily to standardize and compare the
numerous mAbs described by different groups putatively
identifying the same antigen. For example, the TD-4
workshop compared the 56 different mAbs against the
MUC1 mucin (the international antigen designation for
CA 15-3 or CA 27.29). The majority of the antibodies
(34/56) apparently react with the 20 amino acid tandem
repeat sequence of the core peptide of MUC1 mucin
(TAPPAHGVTSAPDTRPAPGS). Many of the remain-
ing antibodies react with carbohydrate epitopes. This type
of analysis at least takes the ﬁrst step toward a blind com-
parison of the various antibodies, all of which are puta-
tively measuring the same antigen with various afﬁnities
and overlapping epitope speciﬁcities. It is not surprising
that the clinical sensitivities and speciﬁcities of breast
immunoassays constructed using the various antibodies are
essentially similar, but some may exhibit unique or subtle
abilities to be clinically more useful than others.
In the remainder of this chapter, the analytes generally
regarded as being the most clinically useful are discussed in
detail. Unfortunately, an exhaustive survey of all the can-
cer markers utilized in various continents is not possible in
this review, as also the numerous tumor markers and their
assays described in the research literature (see references).
A host of new analytes has appeared on the commercial
scene in the last few years, and the clinical utility of many
of these needs to be further explored.
Two new developments that are likely to open new vistas
in cancer research need to be addressed. One is the emerg-
ing ﬁelds of genomics and proteomics that have ushered in
a new era of diagnostic possibilities. Several new cancer-
associated/speciﬁc genes and corresponding assays have
been developed. A new paradigm of understanding the
total proteome ﬁngerprint patterns is underway, and
serum/plasma is the largest repository of the low and high
abundant proteins. Human serum is estimated to contain
30,000 proteins, and six proteins—albumin, immunoglob-
ulin G, alpha-1-antitrypsin, transferrin, immunoglobulin
A, and haptoglobulin—account for ~85% of the bulk. New
techniques are being developed including gene and pro-
tein array technology coupled with sophisticated mass
spectrometry procedures to detect the disease-speciﬁc/
associated markers. A new ovarian cancer test based on an
algorithm of reading proteomic fragmentation patterns
has been described with the claim of 100% accuracy. In
2003, the FDA approved the ﬁrst commercially available
bladder cancer-detection blood test, based on proteomics
technology, called the BladderChek®. Several new groups
and companies are developing multiplexed immunoassays
based on microarray and nanoarray platforms. It is likely
that such assays for multiple analytes may be approved for
routine use in the future.
Several new cancer therapeutic biotechnology medicines
have been approved based on the understanding of cancer
markers. These include monoclonals as cancer therapeu-
tics such as Herceptin® for breast cancer-associated
marker HER-2/neu, Rituximab® for B-cell cancers, and
the intense research and development on CA-based thera-
peutics, often referred to as cancer vaccines. Cancer mark-
ers have come a long way since their ﬁrst discovery nearly
150 years ago and could be the basis of dominant treatment
strategies in the future.
The discovery of CEA and AFP, half a century ago, ush-
ered in a renewed interest in human tumor markers. The
development of radioimmunoassay technology for insulin
a few years earlier had set the stage for the emergence of
new noninvasive techniques to aid the cancer patient.
Thus, CEA and AFP are considered classical tumor
839CHAPTER 9.13 Cancer Markers
markers. Gold and Freedman (1965), in their landmark
experiment, immunized rabbits with extracts of human
colon cancer tissue. The resulting rabbit antiserum was
absorbed with extracts from normal human gut tissue, and
the enriched polyclonal antibody obtained reacted speciﬁ-
cally to cancer tissues and their extracts. Because the anti-
gen identiﬁed was also found in embryonic tissues, the
term CEA was introduced. Both CEA and AFP are mem-
bers of the family of oncofetal antigens that are normally
expressed only in any quantity in embryonic development
but are also found in adult neoplastic tissues. CEA is one of
the most widely used tumor marker immunoassays with
sales of $50–100 million worldwide.
CEA is a heavily glycosylated cell-surface glycoprotein
and one of a large family of related molecules belonging to
what is now fashionably called a superfamily, which also
includes immunoglobulins. This general classiﬁcation is
based on the degree of similarity between the domains of
different proteins. Nearly 36 different glycoproteins have
been identiﬁed in the CEA family, and they appear to be
derived from 10 genes localized on chromosome 19 in two
clusters. CEA is a non-mucinous, 180kDa glycoprotein
secreted by the epithelial cells of the digestive tract in the
normal fetus and in adult cancers. It exhibits
β-electrophoretic mobility and contains 60% carbohy-
drate by weight, constituting N-acetylglucosamine, man-
nose, fucose, galactose, and sialic acid. The oligosaccharide
chains are approximately 80 in number, linked to the poly-
peptide core by asparagine-N-acetyl glucosamine linkages
(this is termed an N-linked oligosaccharide core in con-
trast to the serine or threonine-O-linked core typical of
mucins) with a high proportion of branched oligosaccha-
ride chains. Although CEA has a high carbohydrate con-
tent, due to the composition of the sugars and the
predominant N-linked oligosaccharide chains, it is not
considered a typical mucin like CA 19-9 or CA 15-3. Con-
siderable heterogeneity exists in CEA preparations from
various sources, and this is probably due to variation in the
In contrast, the polypeptide chain is fairly consistent
between different preparations of CEA. The single protein
chain consists of approximately 829 amino acids with sev-
eral intra-chain disulﬁde bonds. Monoclonal antibodies
utilized in CEA assays bind primarily to the protein chain
rather than to the oligosaccharides.
A number of molecules with structures similar to CEA
have been discovered. These include normal cross-reactive
antigens (NCA 1 and NCA 2), tumor-extracted related
antigen (TEX), normal fecal antigens (NFA 1 and 2),
meconium antigen (MA), and biliary glycoprotein 1 (BGP-
1). Higher molecular mass forms of CEA have been
reported from some colon tumor extracts. The concept of
organ-speciﬁc CEA has also been proposed.
As with many tumor markers, the function of CEA, and
the reason for its appearance in the serum of patients with
cancer, is largely a mystery. As with mucins, the associa-
tion of CEA with the epithelial cells of the digestive tract
may suggest a protective role in the turnover of the diges-
tive epithelium. It is estimated that 70mg/day of CEA is
normally secreted into the digestive lumen to eventually
end in feces, and its appearance in blood is presumed to be
due to a reversal or loss of the normal polar secretory func-
tion of the epithelial cells (Pannall and Kotasek, 1997).
Typically, 2.5ng/mL is used as an upper limit for normal
nonsmokers and 5ng/mL for smokers.
The World Health Organization (WHO) made avail-
able the ﬁrst International Reference Preparation for CEA
(73/601). One International Unit of this standard is equiv-
alent to 100ng of CEA glycoprotein.
CEA is one of the most widely used tumor markers in
oncology today, now surpassed only by PSA. Despite its
widespread use, it is not suitable as a screening test for
asymptomatic people, nor is it a reliable diagnostic test in
patients with symptoms that may be due to cancer. This is
because of the considerable incidence of false positives and
false negatives. However, the presence of carcinoma is
strongly indicated in patients with elevated values, and
CEA is often a very useful test as part of the multiparamet-
ric diagnosis of cancer. The most signiﬁcant use of CEA
assays is in the management of cancer patients by serial
monitoring to determine the following:
G the recurrence or metastatic spread of cancer after ﬁrst-
G the presence of residual or occult metastatic cancer;
G the effectiveness of therapy; and
G the prognosis and staging of patients, when used
with other additional information in colorectal and
lung cancer. Most colorectal patients with preopera-
tive CEA in excess of 20 ng/mL would manifest
recurrence within 14 months after surgery (Wu and
Although CEA is primarily associated with colorectal can-
cers, other malignancies that can cause elevated CEA con-
centrations are those arising from the lung, breast,
stomach, ovary, pancreas, and other organs. A number of
benign conditions may also be responsible for CEA levels
signiﬁcantly higher than normal. These include inﬂamma-
tory diseases of the lung and gastrointestinal (GI) tract and
benign liver disease. Heavy smokers, as a group, also have
an elevated range of CEA values.
However, the most useful clinical application of CEA
analysis is as a noninvasive test for the recurrence of
colorectal cancer. This is particularly diagnostic in patients
whose postoperative levels initially decrease to a normal
level within 6 weeks. CEA concentrations are signiﬁcantly
elevated when the liver is the metastatic site for a primary
colorectal cancer. Patients with elevated preoperative lev-
els of CEA that fail to reduce to normal after the ﬁrst-line
therapy are suspected of having residual disease or occult
cancer. In all these patients, the rise or fall of CEA values
generally reﬂects progression or regression of disease as a
function of the therapeutic treatment.
CEA can be used to stage disease and estimate the prog-
nosis. A good correlation exists between preoperative CEA
values and increased risk of recurrence of disease,
840 The Immunoassay Handbook
particularly in Dukes’ C stage of colorectal cancer. Fully
differentiated colorectal cancer tends to secrete CEA copi-
ously compared to undifferentiated tumors, which are
associated with low levels or do not express the antigen.
Elevated CEA levels are also common in breast and lung
cancer patients with disseminated disease. Increases in
CEA values are usually not apparent in localized or pri-
mary disease. The proﬁles of CEA values in patients being
treated for metastatic breast cancer appear to correlate
well with the therapeutic effectiveness.
G Most of the immunoassay tests for CEA are reasonably
well correlated. However, it is important to note that
the different antibodies utilized have subtle differences
in their afﬁnity for CEA and in their cross-reactivities
to CEA-like material. Hence the switching of immuno-
assay kits during the course of monitoring a single
patient is usually not recommended.
G CEA has a low clinical sensitivity and speciﬁcity as a
tumor marker and is hence not recommended for
screening. Clinically, an elevated CEA value is in itself
not of diagnostic value as a test for cancer and this
parameter should only be used in conjunction with
other clinical observations and diagnostic parameters.
Some patients with colorectal cancer do not exhibit
elevated CEA values and elevated CEA levels in some
patients do not change in accordance with progression
or regression of disease. CEA values can be elevated in
a number of benign conditions.
G Smokers constitute a distinct group with a higher range
of baseline values.
Assays for CEA employ the sandwich (immunometric)
assay principle with either an enzyme or non-enzyme label
as the signal generation method. Most immunoassays for
CEA utilize a pair of monoclonal antibodies or a combina-
tion of a polyclonal capture antibody with a monoclonal-
Types of Sample
Serum or EDTA plasma.
Frequency of Use
The Russian scientiﬁc group led by Abelev in 1963 dis-
covered the presence of AFP in adult mice with hepato-
mas (liver cancer). The protein is abundantly present in
the fetus, and levels decline rapidly after birth. This
important milestone in the history of tumor marker
oncology resulted in the emergence of the concept of
oncodevelopmental or oncofetal antigens as possible
tumor markers. The notion of regarding a tumor or can-
cerous state as simulating the fetal or ontogenic pheno-
type subsequently emerged. Many other oncofetal tumor
markers have since been described including CEA and
other cell surface glycoconjugates. The dedifferentiation
of the adult cells and tissues expressing these early embry-
onic antigens suggests that they may be involved in cell
division and the regulation of growth.
AFP is a 70kDa glycoprotein with a single polypeptide
chain. It is similar to serum albumin in size, structure, and
amino acid composition but has distinct immunological
properties. Unlike AFP, however, albumin is not a glyco-
protein. A fucosylated form of AFP has been identiﬁed as
being associated with liver cancer but not in benign liver
diseases. AFP is synthesized by the liver, yolk sac, and GI
tract of the fetus, reaching a peak serum concentration of
up to 10mg/mL at 12 weeks of gestation. This peak level
gradually decreases and, 1 year after the birth of the new-
born, the serum levels decrease to less than 25ng/mL.
Albumin becomes the major serum component in adult
serum with concentrations up to 60mg/mL.
AFP is one of the major components of fetal serum and is
replaced by albumin postpartum. Both of these proteins
are known to be responsible for the maintenance of serum
osmotic pressure and to have various transport functions.
One hundred per cent of healthy males and 97% of healthy
nonpregnant females have AFP values less than 15ng/mL
(Abbott AFP EIA).
AFP determinations are used primarily in two areas. The
application of this test in the detection of open neural tube
defects in the fetus is described elsewhere (see PREGNANCY).
In Asia, due to the high prevalence of liver cancer, particu-
larly in the hepatitis and cirrhosis groups, AFP has been
successfully used in screening applications.
The other common application in the ﬁeld of cancer is
in the management of germ cell tumors and hepatomas.
The most common testicular malignancies derive from
seminiferous tubules and germ cells. They are classiﬁed
into two groups, namely seminomas and non-seminomas.
The non-seminomatous group includes the embryonal
carcinomas, teratocarcinomas, and choriocarcinomas.
The magnitude of AFP elevation has been found to cor-
relate with the stage of non-seminomatous testicular can-
cers, particularly in the embryonal carcinoma group. For
example, continued AFP elevations following orchidec-
tomy (removal of a testis) suggest that the disease is at
stage II or beyond. When sequential monitoring gives rise
to a proﬁle of continued elevations or a rise in AFP levels,
residual disease or a recurrence is strongly suggested.
Decreases in AFP levels in patients are associated with
clinical remission. hCG is often used in conjunction with
AFP to monitor testicular cancers, particularly non-semi-
nomatous tumors of the choriocarcinoma type (see HUMAN
AFP levels are elevated in more than 60% of liver can-
cers. Hepatomas are not common in the western world but
are more prevalent in Africa and Asia. A strong etiological
association between hepatomas and viral hepatitis, other
841CHAPTER 9.13 Cancer Markers
infections, and aﬂatoxin poison ingestion, has been
observed. Clinically, the response to treatment and hence
prognosis of patients with liver cancer has been poor to
date. Nevertheless, AFP determinations have been useful
in monitoring these patients during the course of their
treatment. A few AFP screening programs for hepatomas
have been conducted in high-risk populations with consid-
G AFP levels are known to be elevated in a number of
benign diseases and conditions including pregnancy
and nonmalignant liver diseases such as hepatitis and
G Although limited screening studies have been con-
ducted with promising results, AFP is neither recom-
mended as a screening test or as a diagnostic test.
Immunometric assays are used for AFP determinations.
These normally use a pair of monoclonal antibodies or a
polyclonal capture antibody with a labeled monoclonal
Types of Sample
Serum or plasma (and amniotic ﬂuid for pregnancy
Frequency of Use
CARBOHYDRATE ANTIGEN 19-9
Carbohydrate antigen 19-9 (CA 19-9) or sialyl Lewisa is a
tumor marker predominantly associated with pancreatic,
gall bladder, gastric, and colorectal cancers, which are col-
lectively classiﬁed as GI malignancies. The term GI can-
cer-associated antigen (GICA) has also been used, though
less frequently, to identify the same antigen. The antigen
was originally described as a cell surface monosialogangli-
oside isolated from the SW1116 human colorectal carci-
noma cell line, grown in vitro. The antigen has the chemical
structure shown in Fig. 4 along with other related tumor
The original hybridoma secreting the mAb 1116
NS-19.9 used to characterize the ganglioside antigen was
developed by immunizing mice with the SW1116 human
cancer cells. The minimal structure recognized by this
antibody, and several other antibodies developed subse-
quently, appears to be the terminal tetrasaccharide of the
CA 19-9 antigen. Deletion of the sialic acid moiety or the
fucose residue abolishes or greatly reduces the antigen–
antibody interaction. The ﬁrst comparative study (TD-6
Workshop) of 20 monoclonal antibodies against sialyl
Lewisa and related antigens was completed by Rye et al.
(1998). Cross-reactivities to closely-related oligosaccha-
rides such as sialyl Lewisx, Lewisa, Lewisx, LSTa (CA 50)
and others (Fig. 4) were studied. Most antibodies reacted
to the sialyl Lewisa antigen and exhibited varying degrees
of cross-reactivities to related structures. The subtle dif-
ferences in their cross-reactivities and afﬁnities, together
with the class of the antibody (bivalent IgG versus decava-
lent IgM measuring polyepitopic mucinous antigens),
could explain the spectrum of clinical results obtained.
Claims of superior sensitivities and speciﬁcities have been
made for the various immunoassays even though these are
recommended only for monitoring and not for screening
The CA 19-9 antigen was initially found to be present
in serum from patients with GI malignancies but not in
normal sera. Based upon these ﬁndings, it was hypothe-
sized that the ganglioside antigen was shed into the
serum. However, a more detailed investigation revealed
that the circulating antigen was a high molecular mass
mucinous antigen. Virtually no ganglioside antigen was
found in the serum of cancer patients. Several other forms
of sialyl Lewisa antigen have been described from seminal
plasma, normal saliva, and human milk. A sialyl Lewisa
hexasaccharide with a reducing end (i.e., without the
ceramide of the CA 19-9 ganglioside) has been puriﬁed
from human milk. Thus, it is now established that multi-
ple species of CA 19-9 antigens exist with univalent
(monosialoganglioside, hexasaccharide), oligovalent, or
polyvalent (glycoproteins and mucins) expression of sialyl
The mucinous form of the antigen has been further
characterized following puriﬁcation. The subunit struc-
ture of the mucin appears to be a 210kDa glycoprotein
which, in the absence of detergents or other dissociating
conditions, aggregates to form higher relative molecular
mass species in the range of 600–2000kDa. More than
85% of the glycoprotein is carbohydrate by weight. About
35% of the core protein is composed of serine, threonine,
and proline, a feature typical of epithelial tumor-associated
FIGURE 4 Carbohydrate tumor and related antigens.
842 The Immunoassay Handbook
The CA 19-9 antigen is a sialated derivative of the Lewisa
blood group antigen. The speciﬁc function of such anti-
gens is largely unknown although a number of theories
have been put forward. Gangliosides are thought to be
involved in interactions between cells. Mucins derived
from epithelial cells may have a protective role. Milk oli-
gosaccharides appear to have a bacteriostatic effect.
Typically a reference interval of 0–37U/mL is used (this
may vary according to the method). The arbitrary unit of
antigen has been given a gravimetric value of 0.89ng.
In a signiﬁcant number of GI malignancies, CA 19-9 levels
are elevated above the 37U/mL level. This is particularly
pronounced in pancreatic and gall bladder cancer patients,
followed by gastric and colorectal cancer patients. Like all
tumor marker assays currently available commercially, the
clinical sensitivity of the CA 19-9 marker is moderate in
early-stage disease. The important feature of CA 19-9
assays is the high speciﬁcity of the test. Less than 1% of
apparently healthy blood donors exhibit elevated values. A
number of benign conditions related to GI disease have
been tested for the presence of the antigen, and although
levels are higher than the healthy blood donor group, they
tend to be much less elevated than those samples from can-
cer patients that give a positive result in the test.
CA 19-9 levels are found to be remarkably high in symp-
tomatic pancreatic and gall bladder cancers. The mean
serum level for these cancers is 10–100 times higher than
those for gastric and colorectal cancers. Because benign
gall bladder disease, pancreatitis, and benign hepatobiliary
conditions can frequently cause CA 19-9 levels above the
range for healthy individuals, it is helpful to use an elevated
cutoff level for cancers of the pancreas and gall bladder.
The main clinical application of CA 19-9 determina-
tions is in the monitoring of pancreatic cancer patients.
CEA is usually preferred to watch colorectal cancer. CA
19-9 can also be elevated in other forms of digestive tract
cancer, especially cancers of the stomach and bile ducts
and in some noncancerous conditions such as thyroid dis-
ease, inﬂammatory bowel disease, and pancreatitis (inﬂam-
mation of the pancreas).
G One of the most important limitations of CA 19-9
determinations is the particular sensitivity of the assay
and the tumor marker to viral and bacterial neuramini-
dases resulting in false negatives. Serum samples should
be carefully prepared to avoid bacterial contamination.
G The Lewis blood group antigens are classiﬁed into
Lewisa (approximately 40%), Lewisb (40%), Lewisab
(15%), and Lewisa−b− (5%). CA 19-9 antigen is not syn-
thesized in individuals who are genotypically Lewisa−b−
because of the lack of the enzyme fucosyl transferase.
G The distribution of antigen levels in normal donors
may vary because of the population distribution of
Lewis genotypes in a given geographical area. Most
manufacturers recommend that establishment of cutoff
values is determined by the clinical laboratory.
G Nonlinear dilution, with increased recovery of the anti-
gen, is common in immunoassays for mucins. This is
probably due to a variety of factors such as the presence
of high levels of anti-carbohydrate antibodies in serum,
which generate complexes, the inherent property of
mucins to aggregate and disaggregate into a range of
molecular species, and other matrix-related effects
G Elevated levels of CA 19-9 can be found in some benign
conditions such as cirrhosis and other liver diseases,
gall bladder disease, pancreatitis, and cystic ﬁbrosis,
thus limiting the diagnostic utility of the marker.
Most of the kits developed for CA 19-9 utilize immuno-
metric (sandwich) assay methodology, although one kit
(TRUQUANT® GI™ RIA) is based on competitive inhi-
bition assay. The sandwich assay format utilizes the same
antibody for capture and signal generation. Thus, the CA
19-9 species measured by this homo-sandwich technology
needs to be oligovalent or polyvalent for sialyl Lewisa. The
competitive assay for CA 19-9 uses a solid phase coated
with CA 19-9 and has the potential to measure all species
of CA 19-9 irrespective of the valency for sialyl Lewisa.
Types of Sample
Serum or plasma. Some assays are validated only for serum
Frequency of Use
CANCER ANTIGEN 125 (MUC16)
CA 125 is the most important cancer-associated marker
for the management of ovarian cancer. It was discovered
using a monoclonal antibody, OC125, generated by immu-
nizing a mouse with a human ovarian cystadenocarcinoma
cell line. This antibody exhibits speciﬁcity for staining epi-
thelial ovarian carcinoma cell lines and tumor tissues. The
CA 125 antigen is also expressed in a number of gyneco-
logical, non-ovarian, and normal tissues of Müllerian ori-
gin. Several other monoclonal antibodies have been
developed subsequently to measure CA 125 antigen, and a
comparative blind evaluation was the subject of the TD 1
workshop. These CA 125 antibodies appear to cluster into
two major epitope groups, namely “OC125” like and
“M-11” like (Nustad et al., 1996). Two newer antigens CA
130 and CA 602 were described that appeared to be CA
125-like. The CA 130 antigen employs 130-22 as the
solid-phase antibody and OC125 as the tracer, while the
CA 602 antigen is measured using the two anti-clear cell
ovarian cancer Mabs MA602-1 and MA602-6.
CA 125 is now known as mucin 16 or MUC16 as it is
encoded for by the MUC16 gene. MUC16 is a large,
membrane-associated glycoprotein containing about
22,000 amino acids (3–5 million Da on average, in epi-
843CHAPTER 9.13 Cancer Markers
The CA 125 antigen is expressed in copious amounts in
the tissues and serum of epithelial ovarian carcinoma
patients. The antigen is minimally present in normal
serum, or in adult or fetal ovaries. Using immunohisto-
chemical techniques, CA 125 reactive material has been
detected in some normal tissues such as adult pleura, peri-
cardium, peritoneum, Fallopian tubes, endometrium, and
endocervix. The antigen is also found in the chorionic
membrane, extracts of maternal decidua, and, abundantly,
in the amniotic ﬂuid. Amniotic ﬂuid CA 125 has two dis-
similar subunits and is not derived from the fetus. It is
present in the cornea, conjunctiva in the eye, and the respi-
ratory tract and female reproductive tract epithelial cells.
The high level of glycosylation creates a hydrophobic
environment that acts as a lubricating barrier against for-
eign particles and infectious agents.
Tests for CA 125 typically adopt a 35U/mL discrimina-
tion value that encompasses 99% of healthy donors.
The measurement of CA 125 antigen is very helpful in the
management of serous ovarian carcinomas. Epithelial
ovarian carcinomas frequently metastasize into the perito-
neal cavity on the serosal surfaces, often producing ascites.
Primary ovarian cancer is usually treated by surgically
removing the ovaries and giving the patient chemotherapy
to ablate any residual disease. CA 125 antigen measure-
ment is used to monitor residual tumor burden in patients
who have undergone such therapy. Antigen levels above
the normal range are usually predictive of residual or
recurrent ovarian carcinoma if other causes of CA 125
elevations can be eliminated (see LIMITATIONS). This
intended clinical use of the CA 125 antigen assay was
approved by the US FDA in 1986, and its routine use has
had a strong positive impact in the management of epithe-
lial ovarian cancers. Subsequently, numerous clinical
reports have appeared in the literature suggesting the
extension of the use of CA 125 immunoassays for a variety
of other oncological applications such as limited diagnosis
of ovarian cancer, monitoring of ovarian, lung and breast
cancer patients, and in applications involving endometrial
and fallopian tube cancer. Some studies have attempted to
establish that CA 125 could be used to screen for ovarian
cancer, using a higher cutoff value than the usual 99%
conﬁdence interval for normals. But because ovarian can-
cer is relatively rare, and increased CA 125 levels due to a
range of causes are not uncommon, this test is considered
to be unsuitable for screening.
G CA 125 antigen levels are elevated above the recom-
mended cutoff value in 1% of normals, 5% of benign
diseases, and 28% of non-gynecological cancers. The
benign conditions associated with increased CA 125 in
serum include ovarian cysts, severe endometriosis,
menstruation, ﬁrst trimester of pregnancy, cirrhosis,
G Higher CA 125 antigen levels are also found in non-
ovarian tumors such as those originating in the breast,
lung, uterus, endometrium, pancreas, and liver. Some
early attempts were made to use CA 125 to classify
unknown cancers as being from an ovarian primary.
However, the presence of elevated CA 125 antigen lev-
els in non-ovarian cancers limits the potential of such
G Increased CA 125 antigen levels are found in a number
of ascites ﬂuids and pleural effusions in both malignant
and benign conditions. In our experience, we have
found CA 125 levels in these ﬂuids in the range of
G Radiolabeled OC125 antibody has been used to identify
cancer sites in vivo often missed by other diagnostic meth-
ods. The injection of the mouse antibody into humans
elicits a human anti-mouse antibody (anti-isotypic and
anti-idotypic HAMA) response, capable of increasing the
apparent CA 125 concentration in subsequent serum
samples tested in immunometric assays. Assays employ-
ing alternative antibodies may be used in these situations.
G As with CA 19-9, increased recovery of antigen can occur
in dilution experiments. Considerable caution should be
exercised when carrying out comparisons between meth-
ods or changing from one method to another.
CA 125 assays are based on immunometric (sandwich)
Types of Sample
Serum or plasma. Some assays are validated only for serum
samples. Ascites and pleural effusions should not be used
as they have higher antigen levels than are found in serum.
Frequency of Use
CANCER ANTIGENS 15-3
AND 27.29 (MUC1)
The breast cancer-associated antigen CA 15-3 is a large
mucinous glycoprotein with a native molecular mass in
excess of 400kDa. The antigen is identiﬁed using a sand-
wich (immunometric) assay employing two monoclonal
antibodies. The solid-phase mAb 115D8 was generated by
immunizing mice with defatted human milk fat globule
(HMFG) antigens. The tracer mAb DF3 was developed
against enriched antigens from the membrane of human
breast carcinoma metastasis. mAb DF3 is more speciﬁc for
cancers than 115D8. The sandwich assay that uses these two
antibodies detects antigens that have been variously described
as MAM6, milk mucin, human mammary epithelial anti-
gen, HMFG antigen, and polymorphic epithelial mucin.
The ﬁrst international workshop on cancer-associated
mucins assigned the name MUC1 to the breast cancer-
associated mucin. It is expressed by the MUC1 gene. The
antigen identiﬁed by the DF3 antibody in human milk con-
sists of a single high-molecular-mass species, whereas in
breast carcinomas, the antibody binds to two glycoproteins
844 The Immunoassay Handbook
with molecular masses of 330 and 450kDa. Approximately
50% of the composition by mass is carbohydrate. The anti-
genic site identiﬁed by DF3 appears to be sensitive to neur-
aminidase, alkaline borohydride treatment, and proteases,
suggesting that it is a combined sialyl oligosaccharide and
peptide on the CA 15-3 antigen. Microheterogeneity and
genetic polymorphism are observed in these epithelial anti-
gens causing considerable variation in the size of native
oligomers, subunits, and core proteins from different
sources. Recently, a 309 base pair cDNA, encoding the
sequence for the DF3 antigen, has been isolated, and using
this probe, it was demonstrated that the polymorphism of
these mucins is a reﬂection of the variations in the size of
the alleles. The conserved sequence is rich in guanine and
cytosine with a 60 base pair tandem repeat encoding a ser-
ine-, threonine-, and proline-rich polypeptide. The num-
ber of tandem repeats of this 20 amino acid sequence
(PDTRPAPGSTAPPAHGVTSA) is thought to be the
basis for the polymorphism in these mucins. This peptide
has been synthesized without any oligosaccharide chains,
and at high concentrations, it can block DF3 binding to
solid-phase mucin antigen.
A number of other antibodies have been prepared that
apparently react with the same family of polymorphic epi-
thelial mucins (e.g., CA 27.29, CA 549, MCA, etc.). These
56 mAbs were investigated in the TD-4 workshop by 16
international groups (Price et al., 1998). Most of the mAbs
(34/56) were mapped within the immunodominant
20 amino acid tandem repeat domain. The bulk of the
remaining antibodies appear to recognize carbohydrate-
CA 27.29 is routinely used although it does not appear
to be any better than CA 15-3 in terms of clinical sensitiv-
ity, but it may be more speciﬁc to cancers, i.e., it is less
likely to be positive in patients without cancer.
MUC1 is found in normal and cancerous epithelial cells
and, as a mucin, it is often assumed to play a protective
role. The antigen constitutes approximately 15% of the
total membrane protein of HMFGs. The quantity of DF3
antigen expressed appears to correlate with the degree of
breast cancer differentiation. Because human milk also
contains the antigen, the DF3 antigen is considered a
marker of differentiation of mammary epithelial cells.
Typically, the normal range for CA 15-3 is considered to
be less than 30U/mL in the Centocor CA 15-3 RIA kit.
But women without cancer may have levels as high as
The normal level for CA 27.29 is usually less than
The CA 15-3 antigen is an epithelial membrane antigen
expressed on normal cells and found in serum. Elevated
levels of this antigen are found in about 60% of preopera-
tive breast cancer and 80% of advanced metastatic breast
cancer. Breast cancer is one of the most common cancers
in women in the western world, and the CA 15-3 assay has
proved to be helpful in patient monitoring, with better
clinical sensitivity than CEA assays. An advantage over
CEA is that the antigen levels are not abnormally elevated
in smokers. The CA 15-3 assay is not suitable as a diagnos-
tic test because of its low sensitivity in stage I and II dis-
ease, but in advanced mammary carcinomas, trends in the
antigen levels provide a useful noninvasive indicator of
early recurrence, presence of residual disease, and contin-
ued remission or poor prognosis. Combined use of CA
15-3 and CEA does not appear to give any improved clini-
Elevated levels are found in less than 10% of patients
with early disease and in about 70% of patients with
advanced disease. Levels usually fall if treatment is success-
ful, but initially the levels can rise as dead cells release their
contents into the blood.
CA 27.29 is no better at detection of cancer at any stage
than CA 15-3 but may be less likely to be positive in
patients without cancer. It can be detected in other cancers
and some noncancerous conditions.
G CA 15-3 is only elevated in 10% of patients with early-
stage breast cancer.
G CA 15-3 is sensitive to proteases and neuraminidases,
and hence it is important to prepare and store samples
with great care to avoid microbial contamination.
G Levels of CA 15-3 can also be elevated due to lung and
G Elevated values are seen in less than 10% of benign dis-
eases of liver, breast, ovary, GI tract, and lung.
G The polymorphic, glycoprotein structure of MUC1,
detected by CA 15-3 and CA 27.29, presents similar
assay problems (e.g., dilution nonlinearity) to those
described for other mucins such as CA 19-9.
G CA 27.29 is not elevated in all patients with breast
G CA 27.29 may be elevated in some noncancerous and
cancerous conditions other than breast cancer.
The TRUQUANT BR RIA is a competitive assay with a
mucin-coated solid phase and was the ﬁrst breast cancer
MUC1 marker test approved by the FDA in 1995.
Subsequently, the reclassiﬁcation of tumor markers as class
II devices by FDA allowed other similar immunoassays
to be approved as well. CA 15-3 assays are usually
immunometric, utilizing two different monoclonal anti-
bodies. For example, mAb 115D8 is used as the solid-phase
capture antibody, and labeled DF3 is employed as the sig-
nal generation mAb. The tracer appears to have a more
restricted antigen speciﬁcity than the capture mAb. An
FDA-approved, automated, 15min, luminescence-based
assay for CA 27.29 has also been developed (Siemens
Healthcare Diagnostics, USA).
Types of Sample
Serum or plasma. Some assays may only be suitable for
845CHAPTER 9.13 Cancer Markers
Frequency of Use
Common in Japan, Europe, and the USA.
ESTROGEN RECEPTOR AND
Estrogens are female sex hormones synthesized by the
ovary and adrenal cortex. The hormonal action of estro-
gens is mediated by an estrogen receptor (ER) protein
called estrophilin, which is present in the nuclei of target
cells. It was originally believed that β-estradiol, the major
estrogen, was bound by a cytosolic ER that subsequently
underwent macromolecular size alterations prior to trans-
location into the nucleus to regulate gene expression as a
transcription factor. It is now understood that most, if not
all, of the ER is a nuclear protein with a high afﬁnity for
estradiol. The dissociation constant (kd) is in the range of
10−10–10−9 M. This 66kDa protein has a steroid-binding
site as well as a DNA-binding site. Upon binding of the
steroid, the complex binds to DNA and regulates gene
expression. The estrogen receptors in human breast tissue
generally decrease during the development and onset of
mammary carcinoma. The estimation of ER in breast can-
cer tissues is an important aid in deciding the course of
Progesterone is a steroid hormone that inﬂuences the
endometrium to allow implantation of the fertilized
ovum and its gestation. It is biochemically also a precur-
sor of adrenal corticosteroids, estrogens, and androgens.
The cellular progesterone receptor (PR) has two molec-
ular components with molecular masses of 120 and
95 kDa. Estrogen modulates the appearance of the PR
and its analysis complements the information derived
from ER assays.
The function of ER is to act as the second messenger of
estrogen action by regulating gene expression in the
nucleus. The estrogen–ER complex is capable of stimulat-
ing gene expression by acting on nuclear DNA. Progester-
one upon binding to PR promotes binding and activates
the hormone-speciﬁc genes.
A cutoff of 10fmol ER per mg cytosol protein is recom-
mended for the Abbott ER enzyme immunoassay. The PR
enzyme immunoassay has a cutoff level of 15fmol/mg
Approximately two-thirds of endometrial and breast can-
cers are positive for ER. At least 50% of ER-positive breast
cancer patients respond favorably to endocrine therapy,
while less than 10% of ER-negative patients show such a
good clinical response. Hence, estimation of ER levels has
become fairly routine in determining the choice of therapy
for breast cancer patients. The level of ER also has a prog-
nostic value as there is a good correlation between breast
cancer patients who beneﬁt from endocrine therapy and
the amount of the receptor present in the sample.
In addition to quantitative analysis of ER in breast can-
cer tissues, several oncologists promote the use of direct
visualization of the receptor in tissues by immunohistology
or immunocytology. This method involves sectioning of
tissue or smearing a ﬁne-needle aspirate on a slide fol-
lowed by staining ER-containing cells with a speciﬁc probe
such as an anti-ER antibody. Immunocytochemical meth-
ods reveal the heterogeneity in the tumor with regard to
ER status and make obvious the contribution, if any, from
normal tissue. With this method, it is possible to distin-
guish, for example, between one patient exhibiting ER
positivity in homogenates due to high receptor content in
a small proportion of the tumor and another with moder-
ate amounts of ER in most of the tumor cells. The latter
patient is likely to respond better to endocrine therapy.
Combination of ER and PR estimations appears to
increase the predictive value of those patients likely to
respond to endocrine therapy.
G A biopsy sample is required for the assay.
G Immunoassays for ER (and PR) analysis measure both
the unbound and hormone-bound protein forms,
unlike steroid-binding assays, which use radiolabeled
estrogen or progesterone as tracers. Hence, some dis-
crepancies may be observed between the two types of
The ﬁrst assay method used to identify and measure recep-
tors depended on the binding of 3H-labeled steroids. Such
assays are affected by endogenous steroids, which block
the binding sites, causing an underestimate of the receptor
content. The immunometric enzyme immunoassays, using
monoclonal antibodies to measure ER and PR, introduced
by Abbott Laboratories, appear to be unaffected by endog-
Types of Sample
Homogenate of a biopsy tissue prepared carefully to avoid
heat stress, which destroys the receptors.
For immunocytochemical analysis, fresh or frozen spec-
imens or ﬁne-needle aspirates are required. Parafﬁn sec-
tions are less desirable for the Abbott ER assay.
Frequency of Use
Common for ER, but uncommon for PR assays.
FECAL OCCULT BLOOD
A number of colorectal disorders, benign and malignant,
precipitate the rupture of tissues and blood vessels, result-
ing in the presence of blood in the lumen of the colon and
the rectum. Some of these blood components are found in
the feces, and chemical and immunochemical methods
have been developed to detect their presence. All the com-
mercial tests available are designed to detect hemoglobin.
The chemical tests for fecal occult blood detection are
popularly known as guaiac tests and are based on the
846 The Immunoassay Handbook
pseudoperoxidase activity of heme in hemoglobin. In the
presence of suitable substrates such as gum guaiac (a natural
resin from the wood of Guaiacum ofﬁcinale containing
α-guaiaconic acid) and hydrogen peroxide, the heme cata-
lyzes a peroxidation reaction generating a blue quinone
product. Exploiting this principle, a variety of tests are
available, based on guaiac-impregnated paper or tape,
which can be used to detect fecal occult blood in a labora-
tory, physician’s ofﬁce, or as a home test. The presence of
blood in the stool is a diagnostic aid in the detection of a
number of colorectal disorders including colorectal cancer.
The immunochemical test for fecal occult blood detec-
tion utilizes an mAb that is highly speciﬁc for human
hemoglobin and has a low cross-reactivity with hemoglo-
bins from common dietary meat products. This assay
appears to possess better sensitivity and speciﬁcity than
Fecal occult blood tests are qualitative tests with a positive
or negative end point.
The detection of occult blood in human feces gives a gen-
eral indication of disorders in the colon and rectum and is
not speciﬁc for colorectal cancer. Noncancerous condi-
tions, showing a positive fecal blood test, include peptic
ulcer, ulcerative colitis, and iron-deﬁciency anemia.
Despite these limitations, the qualitative assay for detect-
ing fecal blood was the ﬁrst test used in the western world
as a cancer screening test, with limited success.
Generally, the fecal blood test is recommended as a
diagnostic aid during routine physical examinations of
people above the age of 50. The American Cancer Society
recommends serial testing for three consecutive days to
minimize false-negative results. A special diet is recom-
mended for at least 2 days prior to the chemical test to
avoid false positivity due to any consumption of red meat
or peroxidase-rich vegetables and fruits. Large amounts of
vitamin C in the diet can cause false-negative results.
The immunochemical fecal blood test does not require
patient compliance to the special diet. In an asymptomatic
group, about 2–3% of individuals score positive for fecal
occult blood, of which the incidences of adenomatous pol-
yps and colorectal cancer are 1% and 0.2%, respectively.
The polyps are often considered a precancerous condition.
When used in conjunction with sigmoidoscopy, colonos-
copy, and barium enema, the fecal blood test is a useful and
simple initial test for the detection of colorectal diseases,
G The chemical tests that detect the pseudoperoxidase
activity of hemoglobin are plagued by a variety of
dietary factors. Red meat and peroxidase-rich vegeta-
bles and fruits generate false positives while vitamin C
can cause false negatives. Patient compliance to a
restricted diet is essential to increase the utility of the
test. The immunochemical test does not appear to be
sensitive to the above factors.
G Intermittent bleeding and a lack of homogeneity in the
distribution of blood in the feces can cause a wide varia-
tion in results. Serial testing is therefore often
The chemical test (Hemeoccult®) is based on the genera-
tion of a blue product when hydrogen peroxide is added to
guaiac-impregnated paper and when the fecal smear has
traces of hemoglobin. The development of a mAb reactive
only to human hemoglobin has resulted in an immuno-
chemical assay (Hemeselect™), from Beckman Coulter,
with increased sensitivity and speciﬁcity.
Type of Sample
Frequency of Use
PSA, also known as gamma seminoprotein or kallikrein-3
(KLK3), is a glycoprotein with a molecular mass of 34kDa
with a single polypeptide chain, encoded by the KLK3
gene. Immunologically and biochemically, PSA is distinct
from PAP. PSA is a serine protease (the active site of the
enzyme has a serine residue), and its labile nature could be
partially attributable to its autocatalytic activity. Human
seminal plasma is a rich source of PSA, and histologically,
it is restricted to the cytoplasm of the acinar cells and duc-
tal epithelium of the prostate gland. PSA derives its name
from the observation that it is a normal antigen of the
prostate but is not found in other normal or malignant tis-
sues, although PSA-like material has been recently
described in the breast tissue. The antigen is present in
benign, malignant, and metastatic prostate cancer, and
immunohistochemical analysis of distant metastasis for
PSA can usually identify whether the primary origin of the
cancer is from the prostate.
In serum, at least three forms of complexed PSA have
been identiﬁed in addition to free PSA. One is bound to
alpha2-macroglobulin, and it appears that the PSA epit-
opes are covered and inaccessible for measurement by cur-
rent assays. The second major species is the PSA–ACT
(alpha-1 anti-chymotrypsin) complex. The third complex
is with alpha-1 protease inhibitor (PSA–API). The dis-
crepancy between the various immunoassays for PSA could
be due to their epitope speciﬁcity and the relative ability of
measuring the various species by the different mAbs
employed. Recently, immunoassays measuring total PSA
(often referred to as equimolar assays measuring PSA and
PSA–ACT complex equally well) and free PSA have been
introduced by several diagnostic companies. The research
literature documents numerous novel immunoassays for
the measurement of PSA (see Kreutz and Suresh, 1997)
and the development of two ultrasensitive PSA immunoas-
says deserves special mention (Yu et al., 1997; Ellis et al.,
1997). Both of these novel immunoassays demonstrated
utility by monitoring very low PSA antigen <0.1ng/mL,
847CHAPTER 9.13 Cancer Markers
which was the limit of most clinical assays. In the serum of
patients who have undergone radical prostatectomy, theo-
retically, PSA levels should be zero or very close to it after
a few weeks of surgery. The use of ultrasensitive immuno-
assays for monitoring very early recurrence of metastatic
disease, and potential early second-line treatment, is an
exciting possibility not only for prostate cancer but for the
whole range of clinically relevant tumor markers.
An international workshop (TD-3) on the comparative
properties of 82 PSA antibodies was organized (Stenman
et al., 1999). A signiﬁcant ﬁnding was that nearly 17 of
these cross-reacted with human glandular kallikrein
(KLK2), which shares considerable homology with PSA
(see below). In the light of these observations, studies on
the estimation of PSA in serum, or claims of identifying
PSA in non-prostate tissues, are only credible if the assays
employ monoclonal antibodies speciﬁc to unique PSA epi-
topes not shared by hK2 and other kallikreins. For a com-
prehensive summary of the biology and the clinical
applications of PSA and kallikreins, the reader is directed
to a critical review (Rittenhouse et al., 1998). PSA is now
designated as KLK3.
PSA is a protease whose role is to liquefy semen in ejacu-
late, allowing sperm to swim freely. It is also believed to
play a role in dissolving cervical mucous, allowing sperm
to enter the uterus. It is a member of the Kallikrein family
Ninety-nine percent of apparently healthy donors have
total PSA levels of <4ng/mL (may vary between methods).
Values in benign prostate hypertrophy (BPH) are gener-
ally in the range of 4–10ng/mL, which overlaps with the
levels also seen in malignancy. Total PSA values >10mg/
mL are however more likely due to malignancy. Some
authors have suggested that the ratio of free and bound
PSA be used to discriminate between BPH and prostate
cancer. The PSA–ACT fraction is higher in cancer than in
Prostate cancer is the second most prevalent form of male
malignancy and early diagnosis is the key to a potential
cure. The diagnosis of prostatic carcinoma, like all other
cancers, is done by carrying out a number of procedures in
combination, such as rectal examination, ﬁne-needle
biopsy, chest X-ray, bone scan, and serum PAP tests. The
development of immunoassays to measure serum PSA has
provided a valuable adjunct to the diagnosis and manage-
ment of patients with prostatic cancer. The American
Cancer Society in 1992 recommended the use of annual
PSA tests for screening prostate cancer in conjunction with
digital rectal examination in males above the age of 50.
While this has lead to an enormous interest in the develop-
ment of PSA immunoassays for screening applications, the
oncology community is split on the value of such mass
applications in asymptomatic people. The US Preventive
Services Task Force (USPSTF) does not recommend
screening because most prostate cancer is asymptomatic,
and the treatments involve considerable risks of adverse
consequences for the patient. Serum PSA has been found
to be more useful than PAP because of increased clinical
sensitivity. However, about 5% of patients have increased
PAP but normal PSA levels. For this reason, some experts
recommend that a combined PSA and PAP measurement
is more useful than either one in isolation.
Elevations of serum PSA concentrations above 4ng/mL
are found not only in prostate cancer but also in BPH. The
magnitude of the serum PSA elevation is progressive with
the stage of the disease, and the highest levels are seen in
stage D prostatic cancer with metastatic involvement. PSA
is not useful as a speciﬁc diagnostic test for prostate cancer
because of the elevated values in BPH and attempts have
been made to achieve discrimination based on the mea-
surement of free and total PSA. The percent free PSA, as a
proportion of the total, may be useful. If the free PSA is
less than 10% and the total PSA is above the cutoff, the
risk of cancer is much higher.
Nevertheless, PSA is now a routine test in the manage-
ment of patients who have been conﬁrmed to have prostate
cancer. In this clinical application for monitoring prostate
cancer, PSA is superior to PAP as a reliable tumor marker.
Changes in tumor marker levels correspond to classical
trends (see CANCER MARKERS - INTRODUCTION) in most
cases of prostate cancer. PSA is a good marker for estab-
lishing prognosis in prostate cancer.
G Elevation of PSA above 4ng/mL is not diagnostic of
prostate cancer because benign prostatic hypertrophy
and some benign genitourinary diseases also result in
G Massaging the prostate prior to blood sample collection
can result in transient PSA increases.
G PSA levels may be elevated for up to 2 days after
G About 5% of patients with prostate cancer have elevated
PAP but normal PSA concentrations.
Usually, immunometric assays use monoclonal antibodies.
For example, total and free PSA tests are available from
Siemens for the Centaur®.
Types of Sample
Serum or plasma depending on the assay used.
Frequency of Use
β2-Microglobulin (β2M) is a single-chain aglycosyl protein
composed of 100 amino acids. Its molecular mass is
11.8kDa, and it is now known to be the light-chain com-
ponent of the histocompatibility antigens (HLAs). It is
therefore found on all nucleated cells and is present in high
848 The Immunoassay Handbook
concentrations on the lymphocyte cell surface. This small
protein bears sequence homology with immunoglobulins
and is hence classiﬁed as belonging to the superfamily of
β2M, being a small protein, escapes the glomerular ﬁl-
tration network of the kidneys. Most of what passes the
glomeruli is reabsorbed and catabolized by the cells of the
proximal tubules. A small amount of the protein is detected
in normal urine with elevated levels in patients with proxi-
mal tubular dysfunction.
The normal serum levels of β2M are primarily a reﬂec-
tion of HLA metabolism and turnover. It is estimated that,
on a daily basis, about 150mg of free β2M protein is
secreted into the body ﬂuids. The serum levels are altered
in various benign and malignant conditions, and hence,
β2M is a nonspeciﬁc tumor marker.
β2M is an integral component of the HLA antigen system
and is similar in structure to immunoglobulins. The speciﬁc
role of the protein is not yet deﬁned, but as part of the histo-
compatibility complex, it is thought to be involved in molec-
ular recognition, particularly in distinguishing between self
and nonself. The molecule also appears to stabilize the heavy-
chain conformation of the HLA class I molecule, which may
be important in immune recognition and restriction.
Typically, the normal level of β2M is less than 2.5µg/mL
for normal serum and 0.16µg/mL for urine (may vary
Serum β2M levels are elevated in the presence of a number of
solid tumors and lymphomas. However, a variety of nonma-
lignant conditions such as rheumatoid arthritis, AIDS, lupus,
Crohn’s disease, and renal tubular dysfunction cause elevated
levels of the marker. The level of serum β2M also appears to
be an indicator of acute renal transplant rejection.
The role of β2M levels is less certain for solid tumors,
either in monitoring the disease or as an indicator of prog-
nosis. There appears to be a use for this marker in the lym-
phoid malignancies such as Hodgkin’s and non-Hodgkin’s
lymphoma, multiple myeloma, and chronic lymphocytic
leukemia. A high initial level of β2M is an indicator of poor
prognosis and an advanced stage of the disease. It is also
useful for monitoring the course of the disease in these
cancers, particularly in multiple myelomas.
G β2M elevations are not diagnostic of cancer, as a num-
ber of nonmalignant conditions also give rise to ele-
vated concentrations. The changes in β2M levels found
in noncancer conditions include inﬂammatory disor-
ders such as rheumatoid arthritis, Crohn’s disease,
lupus, AIDS, renal tubular dysfunction, and renal
G Although β2M levels are elevated in some solid tumors,
the marker is not useful in prognosis or in monitoring
the disease state in these situations.
Most kits are competitive in design because of the rela-
tively small size of the antigen. Solid-phase polyclonal or
monoclonal antibody-based enzyme immunoassays are
Types of Sample
Serum, plasma, and urine.
Frequency of Use
Not very common, except in Japan.
Enolase is a ubiquitous glycolytic enzyme, which catalyzes
the conversion of 2-phosphoglycerate to phosphoenolpyr-
uvate (see Fig. 5).
The enzyme enolase is also referred to as 2-phospho-D-
glycerate hydrolase or phosphopyruvate hydratase. It is a
dimer that can be composed of three different types of
subunit, namely α, β, or γ. The αα isoenzyme dimer is syn-
thesized by most of the cells in the body and by glial cells
in the brain. This form is sometimes referred to as non-
neuronal enolase (NNE). The β enolase appears to be spe-
ciﬁc to muscle tissue. The γγ and αγ isoenzymes are
collectively referred to as NSE. NSE is produced by nerve
cells or neurons, and neuroendocrine cells, particularly the
cells of the amine precursor uptake and decarboxylation
lineage. NSE is an acidic protein with a native molecular
mass of 78kDa and a subunit molecular mass of 39kDa.
NSE and NNE are immunologically distinct and have dif-
ferent sensitivities to chloride ions and temperature.
NSE is a glycolytic enzyme involved in the energy-gener-
ating process of the cell. Ontogenetically the NSE isoen-
zyme appears in the ﬁnal stages of neuronal differentiation
and is hence a good nerve cell maturation marker.
Abnormal levels are usually higher than 9ng/mL.
NSE is found elevated primarily in small-cell lung cancer
(SCLC) and neuroblastomas. Other neuroendocrine
tumors with elevated levels of NSE include insulinomas,
medullary thyroid carcinomas, pheochromocytoma, and
gut carcinoids. The main clinical application of NSE is in
FIGURE 5 Action of neuron-speciﬁc enolase.
849CHAPTER 9.13 Cancer Markers
the monitoring of these tumors for response to chemo-
therapy or to detect early relapse.
SCLC is the most aggressive of the lung cancers, and
most of the patients have already progressed to metastasis
by the time of diagnosis. However, SCLC responds par-
ticularly well to chemotherapy compared to other lung
cancers. NSE levels can help to classify the type of lung
cancer when used in conjunction with histology, enabling
the appropriate course of therapy to be initiated. Monitor-
ing NSE levels can also assist in determining the effective-
ness of chemotherapy and to predict relapse of the disease.
Neuroblastoma is a common childhood cancer, which is
often malignant. In addition to monitoring, NSE levels
help to differentiate between neuroblastoma and Wilm’s
tumor, which originates in the kidney. Both these condi-
tions may present as a palpable abdominal mass and ele-
vated levels of NSE are suggestive of neuroblastoma.
Chromogranin A appears to be a better marker for car-
Careful specimen handling is essential for this immunoas-
say because of the presence of NSE in erythrocytes and
other blood cells. Avoid samples that are hemolyzed and
exhibiting an absorbance of at least 0.3 at 500nm.
Usually tested using a polyclonal–monoclonal immuno-
Type of Sample
Nonhemolyzed serum samples are required and repeated
freezing and thawing is to be avoided.
Frequency of Use
SQUAMOUS CELL CARCINOMA ANTIGEN
Squamous cell carcinoma (SCC) antigen is a 48kDa glyco-
protein, originally isolated from a squamous cancer of the
uterine cervix. Immunohistochemically, SCC was found to
be a cytoplasmic protein of normal and cancerous squa-
More than 90% of cancers of head and neck cancer and
80% of cervical cancers are SCCs. Studies have indicated
that SCC antigen is a good marker for monitoring the
effectiveness of treatment of SCCs. Although not approved
by the FDA for routine clinical use, the development of a
tumor marker assay for squamous cancers of the head,
neck, lung, and cervix heralds an important step in the
management of these cancers.
A study conducted by ARUP Laboratories established an
upper 97th percentile of 2.2ng/mL (males and females).
SCC antigen is the ﬁrst commercially available tumor
marker for squamous cancers. The serum levels of SCC
antigen are elevated in a signiﬁcant percentage of patients
with squamous cancers of the cervix, head, neck, and lung,
and the level of the tumor marker increases with the stage
of the disease. The speciﬁcity appears to be good for squa-
mous cancers, and adenocarcinomas do not give rise to
abnormal concentrations of this marker. Some benign
gynecological and pulmonary diseases are responsible for
higher SCC values than the normal reference interval.
SCC antigen levels tend to be normal in early-stage squa-
mous cancers. About 40% of stage III and 60% of stage IV
head and neck cancers exhibit SCC antigen levels above
the reference interval for normals. Cervical squamous car-
cinomas in similar stages have shown a higher proportion
(80%) of patients with elevated antigen levels. The degree
of differentiation of the tumor does not appear to be
related to the level of SCC antigen. Monitoring patients
with squamous cancers has demonstrated that the assay
can detect recurrence and provide a prognosis.
Several nonmalignant benign diseases of the skin (e.g.,
eczema) and lungs (e.g., tuberculosis), sarcoidosis, and
other conditions can result in elevated SCC antigen
Currently, the assay methodology employs radiolabeled
SCC antigen and polyclonal antibody in a competitive
Type of Sample
Frequency of Use
Tissue polypeptide antigen (TPA) is a pan-carcinoma
marker. This antigen was discovered in 1957 as an insolu-
ble residue from human carcinomas. TPA is now known to
belong to a class of cytoskeletal proteins called cytokera-
tins or intermediate ﬁlaments. Cytokeratins 8, 18, and 19
react with anti-TPA antibodies. These cytokeratins are
cytoplasmic proteins and are found in all normal epithelial
cells and cells lining the ducts and their sacs. Thus, various
tumors arising from different organ sites are known to
express TPA, which is also released into the serum by cell
destruction. TPA assays represent the ﬁrst-generation
cytokeratin tumor marker tests. CYFRA 21-1 is a second-
generation monoclonal immunoassay, detecting 21-1 frag-
ments of cytokeratin 19.
The cytoskeleton is responsible for the physical three-
dimensional architecture of the cell. During cell
850 The Immunoassay Handbook
division, the cytoskeleton assumes a crucial, dynamic,
and functional role. The precise function of individual
cytokeratins is yet to be fully understood, but as an inter-
mediate ﬁlament, it has an obvious role in deﬁning the
structure of the cytoskeleton and its dynamics during cell
ARUP Laboratories quote a reference interval of less than
2.3ng/mL for CYFRA 21-1. Other methods are likely to
have different reference intervals.
CYFRA 21-1, which measures cytokeratin 19, is elevated
in most lung tumors of the non-small cell category, with
the highest sensitivity in lung squamous cell cancers.
G Cytokeratin markers are not suitable for diagnosis of
carcinoma but are used to monitor patients, often along
with other organ-speciﬁc tumor markers.
G Elevations in TPA are seen in the last trimester of preg-
nancy and in various benign diseases of the lung, liver,
stomach, and pancreas.
G Monitoring of patients with cytokeratin markers during
therapy is more complex than using other markers.
Further work is needed to resolve the nature of these
soluble serum fragments of cytokeratin parent mole-
cules, which are more insoluble in nature.
An enzyme immunoassay is available from Fujirebio Diag-
nostics. The test is also available from Abbott Diagnostics
for the Architect® analyzers.
Type of Sample
Frequency of Use
HUMAN CHORIONIC GONADOTROPIN
See PREGNANCY: HUMAN CHORIONIC GONADOTROPIN for
further information on this marker.
The reference value for pregnancy is typically >25mIU/
mL. However, for the use of beta-hCG for oncology appli-
cations, a cutoff of 5mIU/mL is typical for females and
3mIU/mL for males. This may vary between methods.
hCG is a major analyte in the diagnosis of pregnancy, and
this aspect is covered in a separate chapter. Choriocarcino-
mas and male germ-cell tumors are characterized by ele-
vated levels of hCG and its subunits. Increases in hCG
levels have also been found in cancers of the breast, lung
and small intestine, and in some prostate cancers. The
combined measurement of hCG and AFP levels has been
shown to be superior in conﬁrming diagnosis and manag-
ing non-seminomatous germ-cell tumors. Monitoring of
germ-cell tumors is effective with these markers, which
mirror the clinical progression or regression of the
Cancers secreting hCG often produce abnormal forms
of the molecule. These include altered glycosylation of the
peptide and secretion of α chains. Some scientists have
attempted to develop an hCG assay that is speciﬁc for can-
cerous conditions by exploiting these anomalous features.
This type of assay has not yet become commercially
Types of Sample
Serum or plasma.
Frequency of Use
Not very common as a tumor marker.
HER-2/NEU (HER-2, C-ERB B-2)
The human epidermal growth factor receptor (HER-2)
oncogene encodes a transmembrane tyrosine kinase recep-
tor that is a key indicator marker for invasive breast cancer
and target for therapy. This protein, with other members
of the HER family, acts as a switch that causes cancerous
growth through cell proliferation, motility, resistance to
apoptosis, invasiveness, and angiogenesis. It communicates
molecular signals from outside the cell to the inside, turn-
ing genes on and off. It is elevated in approximately 20%
of breast cancers and, if positive, indicates a poorer prog-
nosis, and that treatment with anti-HER-2 therapy may be
effective. Several therapies, some still in clinical trials, are
aimed at suppressing the activity of the HER family of
proteins, including Herceptin® (trastuzumab), which is a
humanized mAB therapeutic approved for the treatment
of metastatic breast cancer patients with overexpression of
the HER-2/neu growth factor antigen.
The HER-2/c-erb B-2 protein has a critical role in convey-
ing messages into the cell as a member of the growth factor
family, via tyrosine kinase-dependent cascade events.
When an activating ligand binds to this protein, it dimer-
izes by associating with another member of the HER family
or with another HER-2 protein molecule. The speciﬁc
tyrosine residues on the intracellular portion of HER-2
that are phosphorylated, determined by the ligand and
dimerization partner, deﬁne the signaling pathway that is
activated. The wide variety of ligands and crosstalk between
cellular pathways provide a diverse signaling capability.
851CHAPTER 9.13 Cancer Markers
See published guidelines from the American Society of
Clinical Oncology—College of American Pathologists.
If the HER-2 protein is being overexpressed by the tumor,
the prognosis is poorer, and treatment applied to suppress
the HER-2 receptor will be most effective. The measure-
ment of the levels of the HER-2 protein expressed by the
gene is an important marker in the prognosis of breast can-
cer patients. Some stomach tumors are responsive to
HER-2 receptor treatments, and this test may be carried
out on advanced stomach cancers. As with many other
prognostic tumor markers, increased levels indicate poor
prognosis, early relapse, and shorter duration of survival.
See Ross et al., 2009.
Tissue biopsies are examined using slide-based methods
such as immunohistochemistry, ﬂuorescence in situ hybrid-
ization (DNA hybridization using ﬂuorescent-labeled
probes) and chromogenic in situ hybridization. Immuno-
histochemistry is the most commonly used method. For
example, Ventana Medical Systems (Tucson, Arizona)
manufactures the Ventana Pathway™ immunohistochem-
istry assay, and Dako (Glostrup, Denmark) manufactures
the Dako HercepTest™. There are also blood tests using
ELISA (immunometric/sandwich) methodology.
Frequency of Use
This marker is one of the new tumor markers and its clini-
cal use is likely to increase in the future.
BLADDER TUMOR ANTIGEN
Bladder cancer is the sixth most frequent cancer in women
and the fourth most common in men. Men are three times
more likely to get bladder carcinoma than women. Smok-
ing and exposure to chemicals appear to be risk factors.
Most bladder cancers (90%) are transitional cell carcino-
mas of epithelial origin. The rest are squamous cell can-
cers, adenocarcinomas, or undifferentiated carcinomas.
Traditional detection methods are cytoscopy and urinary
cytology, analogous to the PAP smear tests for cervical
cancer. As early as 1945, Papanicolaou and Marshall
described the detection of cancer cells of the urinary tract
by urinary cytospin sediments. Several bladder tumor
marker tests have been developed including bladder tumor
antigen (BTA), urinary cytokeratin, NMP22, and ﬁbrin/
ﬁbrinogen degradation products (FDP).
The BTA antigen appears to be the complement factor
H or a closely-related protein. The apparent molecular
weight of the BTA antigen is predominantly 150kDa,
although some degraded fragments have also been identi-
ﬁed. Several different cancer cell lines have been shown to
secrete BTA into the medium.
The BTA antigen has a complement factor C3b binding
site, and it degrades C3b in the presence of complement
factor I. The structural relationship to serum complement
factor H (hCFH) was deduced by partial sequence analysis.
It is speculated that the secretion of complement factor-
like activities may confer a selective in situ growth advan-
tage to cancer cells by blocking the complement-mediated
lytic activity. The function of hCFH is to interact with
complement factor C3b and inhibit the formation of mem-
brane attack complex, thus preventing cell lysis. The
hCFH appears to have a role in the regulation of the alter-
nate complement pathway.
A cutoff value of 14U/mL has been suggested, based on
the mean plus 3 S.D., derived from urine of healthy indi-
viduals. Some tests are qualitative only.
The BTA antigen is useful in monitoring transitional cell
carcinomas, which represent the bulk of bladder cancers.
The sensitivity is excellent for noninvasive high-grade
tumors and relatively high for low-grade and in situ tumors.
NMP22 does not appear to offer any advantage over
G Urine samples (fresh, refrigerated, or frozen) are used
in this assay, and care should be taken in serial mea-
surements due to variable amounts of voided urine.
G It is not as good as cystoscopy for ﬁnding bladder can-
cer. Many clinicians prefer to use cystoscopy to follow
up bladder cancer treatment.
G BTA antigen is not diagnostic. Renal stones, nephritis,
renal cancer, urinary tract infections, cystitis, or recent
trauma to the bladder or urinary tract can cause false
The BTA TRAK enzyme immunoassay is a dual monoclo-
nal sandwich assay. The same pair of monoclonals are used
in the new qualitative BTA Stat test, which was approved
by the FDA in 2000 as the ﬁrst home-use device for cancer
marker recurrence. This qualitative test is similar to the
lateral ﬂow dipstick pregnancy test based on the principle
of immunochromatography. This test is an adjunct to
cytoscopy, and based on the results of the BTA Stat test,
the urologist has the choice to use either a rigid or ﬂexible
cytoscope. If the results are positive, the rigid cytoscope
could be used under general anesthesia to remove the
recurrent tumor during examination. With a negative
BTA Stat result, the ﬂexible exploratory cytoscope could
be used with only a local anesthetic.
Frequency of Use
The BTA TRACK™ EIA and BTA Stat tests (B.D.S. Inc.,
Redmond, WA, USA) are relatively new assays, and hence,
their use is currently limited. The regulatory endorsement
of the qualitative test may increase its home use in the
852 The Immunoassay Handbook
FOR TUMOR MARKERS
In recent years, there has been a growing number of qualita-
tive or semiquantitative cancer marker assays developed on
the user-friendly lateral ﬂow, immunochromatography for-
mat. This format can accommodate immunometric or com-
petitive immunoassays. In the immunometric format, one of
the antibodies is labeled with either colloidal gold, colored
latex or colloidal carbon to generate the pink, blue, or black
lines, respectively. In the competitive assay format, it is the
analyte that is labeled. The sample moves along the device
by capillary ﬂow, solubilizing the label, and carrying it
across a band of immobilized antibody, on a color-con-
trasted white membrane. A detailed description of several of
these types of assays (for other analyte applications) can be
found in the PRODUCT TECHNOLOGY chapters (Part 7). Also,
see LATERAL FLOW IMMUNOASSAY SYSTEMS. While most of
these types of tests provide qualitative information on the
presence or absence of a given analyte, some degree of
quantitative information is now possible with the recent
development of several instruments developed to measure
the intensity of the end point band. In the future, it is likely
that such tests would be performed in primary health care
centers, such as the physician’s ofﬁce, to initiate appropriate
therapeutic courses of action or referral to secondary and
tertiary health care centers for subsequent follow-up.
Currently a few tumor marker assays are available in the
immunochromatography format. They are:
G Ideal Rapid UBC™ test (IDL Biotech, Sweden)—A one-
step self-testing assay for urinary bladder cancer (UBC)
detecting cytokeratin fragments. A quantitative ELISA
assay format is also available from the same source.
G BTA Stat™ test (B.D.S. Inc., Redmond, Washington,
USA)—An FDA-approved home test for monitoring
recurrence of urinary bladder cancer. This assay mea-
sures the presence of urinary complement factor H and
G One-Step FOB™ test (TECO Diagnostics, California,
USA)—Fecal occult blood test detects the presence of
human hemoglobin in feces. This test utilizes mono-
clonal and polyclonal antibodies speciﬁc to human
hemoglobin and, hence, has less interference from
dietary source hemoglobin, vitamin C, or iron.
G AFP Card™ test (TECO Diagnostics, California,
USA)—This is a one step assay detecting AFP and
could have applications in hepatomas.
G PSA test (PSA-CHECK-1®, PROSTA-CHECK®,
Veda Lab, France)—The 1992 recommendation of the
American Cancer Society to use an annual PSA test in
conjunction with digital rectal examination for every
male above the age of 50 years has likely resulted in the
development of several rapid PSA immunochromatog-
raphy tests. The test from Veda Lab claims to rapidly
discriminate PSA values above or below 4ng/mL, the
accepted cutoff for apparently healthy males.
FREE LIGHT CHAIN ASSAYS
The ﬁrst biochemical cancer marker to be discovered was
the Bence Jones proteins in urine. These are the mostly
monoclonal homogeneous kappa or lambda light chains of
immunoglobulins derived from the malignant B cells.
Until now, the traditional method of detecting free light
chains (FLCs) has been either by electrophoresis of pro-
teins or by immunoﬁxation electrophoresis of urine. The
urinary tests have several limitations such as low sensitiv-
ity, need for stringent urine collection over 24h, subse-
quent concentration prior to assay, and the metabolism of
the light chains by the proximal tubules, thus potentially
masking the malignant condition to some extent. More
recently, serum FLC assays have been developed that are
more sensitive and can be automated (Bradwell, 2003).
FDA-approved immunoassay tests are now available as an
adjunct for the diagnosis and monitoring of multiple
myeloma. The kits are also available for use on two auto-
mated systems (Beckman Coulter IMMAGE® and Sie-
mens Dade-Behring BNII).
The light chain is part of the two-chain immunoglobulin
molecule and is found in all types of immunoglobulin with
some rare exceptions such as camel immunoglobulins, which
have only one chain. The light chain has a constant region
and three variable regions that have unique amino acid
sequences in different antibodies. Together with the three
variable regions of the immunoglobulin heavy chain, the six
unique domains of every immunoglobulin combine to form
the speciﬁc antigen-combining site, called the paratope. The
paratope is the complementary face that binds to the speciﬁc
epitope (antigenic determinant) face on the antigen.
Serum FLC levels are elevated in 85% of patients with
nonsecretory multiple myelomas, 95% of intact immuno-
globulin multiple myeloma, and 100% of light-chain mul-
tiple myeloma. The most important application of the
FLC assays is in the detection of nonsecretory multiple
myelomas when serum or urine electrophoretic tests for
monoclonal proteins are in the normal range. The half-life
of FLC in serum is ~2–4h compared to intact IgG. Hence,
serial measurements of the FLC can be a quicker indicator
of the outcomes of a therapeutic regimen. The serum lev-
els of FLC are also seen in amyloid disease, wherein the
light chains can form polymeric deposits. Decreasing lev-
els of serum FLC as a result of chemotherapy is a very
good indicator of long-term survival.
The immunoassays are based on the development of anti-
bodies speciﬁc to masked unique light chain epitopes. The
developed kits using these antibodies are based on latex-
enhanced nephelometric and turbidimetric methods.
Unlike the classical electrophoretic tests, concentration of
sample is not necessary.
The FLCs are not speciﬁc to cancer, and only 20% of
B-cell chronic lymphocytic leukemias have abnormal
levels. Most monoclonal gammopathies are of undeter-
mined signiﬁcance and 60% have elevated FLC.
853CHAPTER 9.13 Cancer Markers
Frequency of Use
This is a relatively new test and in view of its advantages of
sensitivity and in measuring clinically useful levels in serum,
it is likely to replace the urine-based methods in the future.
Novel Experimental and
Other Minor Markers
This section includes a brief overview of recent immunoas-
says for tumor markers that have not yet achieved wide-
spread acceptance. Many of the antigens have been identiﬁed
by the reactivity of monoclonal antibodies developed by
various investigators. It appears that a number of these
immunoassays determine the levels of novel mucinous anti-
gens. Much remains to be learned about the biochemistry of
these complex molecules, and their utility in a clinical set-
ting is yet to be established. Nevertheless, some of the early
results show promise in the monitoring of cancer patients.
An entirely new class of tumor marker gene-based assays are
emerging, which is beyond the scope of this chapter and
book. Several minor tumor markers, e.g., des-gamma-
prothrombin, calcitonin, ACTH, TA-4, creatine kinase ββ,
inhibin, LDH, TSH, and catecholamines, are used in spe-
ciﬁc clinical niches and are summarized in Table 2.
CANCER ANTIGEN 72.4
Centocor developed this novel panadenocarcinoma muci-
nous marker, utilizing a pair of monoclonal antibodies
CC-49 and B72.3. MAb B72.3 reacts with sialyl 2–6Gal-
NAc-O-Serine/threonine (Sialyl Tn), which is considered
another oncofetal antigen (Fig. 4). This assay appears to be
useful in gastric carcinoma.
The S-100 antigen of neuroendocrine origin is an acidic
protein and is a homo- or heterodimer of A and B sub-
units. The S-100 B serum assay (Sangtec IRMA, Diasorin
AB, Sweden) is a melanoma marker, useful in monitoring
of patients and as a prognostic marker. Elevation of this
antigen in surgically treated and disease-free melanoma
patients suggests early recurrence.
Metra Biosystems (Mountain View, California, USA)
introduced an assay to measure bone ALP. This assay may
be more speciﬁc in identifying bone metastasis, instead of
measuring total ALP, which is also elevated in liver metas-
tasis, hepatomas, and prostate cancer.
The NMP network is the residual framework seen in the
nucleus after exhaustive extraction to remove membrane,
chromatin, and cytoskeletal proteins. The NMP-22 assay
(Matritech, Massachusetts, USA) for bladder cancer iden-
tiﬁes the nuclear mitotic apparatus protein, associated with
the mitotic spindle during mitotis.
Chromogranin A (CgA) is a secretory, acidic protein of the
neuroendocrine granules, of 45kDa. It is elevated in carci-
noid tumors, neuroblastoma, and SCLC. It is abnormal in
one out of three people with these conditions and in two of
three if metastasis has occurred. It can be elevated in some
advanced cases of prostate cancer with neuroendocrine
features. The cutoff is typically 50ng/mL, but it varies
with the method used. See GASTROINTESTINAL TRACT chap-
ter for more detail on this analyte.
Telomeres are the specialized nucleoprotein ends of all
eukaryotic chromosomes, composed of tandem repeats of
the nucleotide sequence TTAGGG. Progressive cell divi-
sions shorten the telomere length, which is thought to be
the biological clock that triggers senescence. However, in
stem cells and cancer cells, this is regenerated by the
enzyme telomerase. Cancer cells appear to escape this
TABLE 2 Minor Tumor Markers Useful in Niche Clinical
Tumor Markers Clinical Application
Hepatoma vs cirrhosis
2. Calcitonin Bone metastasis, meduallary
carcinoma of thyroid
3. Adrenocorticotropic hormone
4. Creatine kinase BB Neuroendocrine tumors
5. TA-4 Squamous cell carcinoma
6. Cancer-associated serum
Mucinous ovarian cancer
7. Inhibin Granulosas and mucinous
8. Lactate dehydrogenase
Germ cell tumors
9. Thyroid stimulating hormone
10. Catecholamines Neuroblastomas, pheochromo-
cytoma, carcinoid tumors
11. Tumor-associated trypsin
Renal and gastric carcinomas
12. Neopterin Prognostic in myeloma and
13. Epidermal growth factor
SCC and prognostic in breast
14. Ferritin Advanced adenocarcinomas
15. 5-hydroxyindoleacetic acid
16. Lipid-associated sialic acid Nonspeciﬁc marker in several
17. Parathyroid hormone-
related peptide (PTH-RP)
Tumors with hypercalcemia
18. Terminal deoxynucleotidyl
Classiﬁcation of leukemias
19. Urinary gonadotropin
854 The Immunoassay Handbook
replicative senescence by upregulating telomerase activity.
In approximately 85% of cancer cells, telomerase activity
is increased, whereas its production is repressed in somatic
cells, except for proliferating progenitor cells and activated
lymphocytes, and hence this is emerging as an exciting
new marker. A PCR-ELISA has been developed by Boeh-
ringer Mannheim, Germany, to measure telomerase activ-
ity. A biotin-labeled speciﬁc primer is elongated by
telomerase and subsequently ampliﬁed by PCR, hybrid-
ized to the telomerase repeat-speciﬁc detection probe. A
streptavidin-coated microtiter plate is used in the ﬁnal
ELISA detection step.
The quantitative UBC antigen is a new test that is useful in
the management of UBC. Unlike most other tumor mark-
ers whose levels in body ﬂuids reﬂect the tumor burden,
the UBC antigen levels are indicative of the course of the
disease measured in terms of tumor cell activity. Hence,
this test is indicated for monitoring tumor recurrences,
and its levels are correlated with the stage and grade of
cancer. Both IRMA and ELISA versions are available for
diagnosis and follow-up, supplemented by a point-of-care
urine dipstick test (UBC® Rapid, IDL Biotech, Sweden).
Normetanephrine and metanephrine are O-methylated
metabolites of the catecholamines, noradrenaline and
adrenaline, respectively. These metabolites are elevated in
pheochromocytoma, ganglioneuroma, and other neuro-
genic tumors. An enzyme immunoassay for the detection
of these two metabolites in heparin plasma has been devel-
oped (DLD Diagnostika GmbH, Germany).
A new cancer marker for acute promyelocytic leukemia
(APL) has been identiﬁed along with the development of a
monoclonal probe (Dako Inc., Denmark) for identiﬁcation
of the promyelocytic leukemia protein (PML). In APL
cells, the nuclei exhibit a microgranular pattern with
numerous small dots, while the normal hematopoietic cells
exhibit the speckled pattern with 5–10 nuclear dots. The
architecture of the PML nuclear granules is distorted in
APL cells, bearing the characteristic reciprocal 15:17 chro-
mosomal translocation. It is important to diagnose APL
from the other types since this leukemia subtype is success-
fully treated often with chemotherapy and all-trans-retinoic
TUMOR M2 PYRUVATE KINASE
Tumor M2 pyruvate kinase was deﬁned by Eigenbrodt
around 1985. In cancers, the active tetrameric form of the
M2 isoenzyme of pyruvate kinase is converted to an inactive
dimeric form by direct interaction with oncoproteins to
channel glucose carbons into DNA synthesis. Circulating
tumor M2 pyruvate kinase is more commonly elevated in
esophageal, gastric, and colorectal cancer patients than con-
ventional tumor markers. Fecal M2 pyruvate kinase is a
sensitive marker of colorectal cancer. As a fecal marker for
colorectal cancers, fecal tumor M2 pyruvate kinase has a sen-
sitivity of 73–92% at a cutoff value of 4U/mL as against 50%
sensitivity for the guaiac fecal test. The marker can also be
measured in plasma. Limited information exists as yet on the
utility of tumor M2 PK as a prognostic marker, as a marker
of malignant transformation, or in assessing tumor recur-
rence or response to treatment. Large multicenter trials are,
therefore, needed to deﬁne its clinical role (Kumar, 2007).
Genes involved in pulmonary carcinogenesis have been
identiﬁed by examining gene expression proﬁles of non-
SCLCs to identify molecules that might serve as diagnos-
tic markers or targets for the development of new molecular
therapies. A gene encoding ADAM8, a disintegrin, and
metalloproteinase domain-8 was one candidate for the
identiﬁcation of such molecules investigated by Ishikawa
and group. Tumor tissue microarray was applied to exam-
ine the expression of ADAM8 protein in archival lung can-
cer samples from patients. Serum ADAM8 levels of 105
lung cancer patients and 72 controls were also measured
by ELISA. A possible role of ADAM8 in cellular motility
was examined by Matrigel™ assays. ADAM8 is abundantly
expressed in the great majority of lung cancers examined.
A high level of ADAM8 expression is signiﬁcantly more
common in advanced stage IIIB/IV adenocarcinomas than
in adenocarcinomas at stages I–IIIA. Serum levels of
ADAM8 were signiﬁcantly higher in lung cancer patients.
Hence, ADAM8 could be useful as a diagnostic marker
and also a therapeutic target.
HE4 is part of a family of protease inhibitors that func-
tions in protective immunity, which is overexpressed in
ovarian cancers, especially in serous and endometrioid his-
totype. It is secreted by the cell and then detectable in the
sera of patients with ovarian carcinoma by an enzyme
immunoassay. Preliminary studies of HE4 suggest a higher
speciﬁcity than CA 125 in different benign and malignant
conditions, excluding renal failure. Patients with renal fail-
ure have very high HE4 serum levels, undistinguishable
from ovarian cancer. For this reason, patients with this
pathology were excluded in our study. Excluding this dis-
ease, slightly elevated HE4 serum levels were found in
only one third of patients with effusions or in 5% of
patients with chronic liver diseases.
Initial validation studies by Roessler et al. conﬁrmed the
relevance of PSME3 as a tumor-associated protein. Poly-
clonal antibody to recombinantly expressed PSME3 was
generated, and upregulation of the protein in colorectal
cancer tissue was conﬁrmed by western blot analysis and
immunohistochemistry. Importantly, the marker could
also be measured in serum using a highly sensitive immu-
noassay and was signiﬁcantly elevated in serum of colorec-
tal cancer patients compared with healthy individuals and
patients with benign bowel disease.
855CHAPTER 9.13 Cancer Markers
The INK4a/ARF locus encodes two unrelated tumor
suppressor proteins, p16INK4a and p14ARF, which par-
ticipate in the two main cell cycle control pathways,
p16INK4a-Rb and p14ARF-p53111–115. p14ARF (p19Arf
in mice) is a 14kDa (19kDa) protein predominantly
localized in the nucleolus. It blocks the cell cycle in both
G1 and G2 phases and inhibits the growth of incipient
cancer cells by indirectly activating p53. It also inhibits
ribosomal RNA processing and interacts with topoisomer-
ase I. Arf triggers sumoylation of many cellular proteins,
including Mdm2 and nucleophosmin (NPM/B23), with
which p19Arf physically interacts in vivo. This occurs
equally well in cells expressing or lacking functional p53.
Thus, Arf’s p53-independent effects on gene expression
and tumor suppression might depend on Arf-induced
Proliferative markers have been broadly evaluated as prog-
nostic factors for early-stage breast cancer patients. Ki67,
a nuclear nonhistone protein, was identiﬁed after immuni-
zation of mice with the Hodgkin’s lymphoma; 10–13 Ki67
is expressed only in cells in the proliferative phases of the
cell cycle (G1, S, G2, and M phases). Ki67 is vital for cell
proliferation, since downregulation of Ki67 using anti-
sense nucleotides prevents cell proliferation. Ki67 is tightly
controlled and regulated, implying a fundamental role in
cell proliferation. However, it has been very difﬁcult to
determine its function because of its lack of obvious
homology with known proteins. Another suggested role of
Ki67 is organizing DNA, based on its localization to extra-
nucleolar sites during early G1; these sites contain centro-
meric and satellite DNA. Ki67 is also known to bind to
DNA. MacCallum and Hall suggested a structural role for
Ki67 within the nucleolus, based on its ability to interact
with other proteins and bind with RNA and DNA. They
also suggested that Ki67 is an essential factor in the syn-
thesis of ribosomes during cell division. Further studies
should be conducted to elucidate the roles of Ki67 in cell
proliferation and tumorigenesis.
VEGF is composed of a family of ﬁve isoforms (VEGFA,
VEGFB, VEGFC, VEGFD, and PLGF) that act as ligands
for tyrosine kinase receptors (VEGF-Rs). Upon binding of
VEGF to its receptors (primarily VEGFR2), intracellular
signaling pathways, including MEK-ERK and PI3K-Akt,
are activated that mediate angiogenic switches. This acti-
vation of angiogenesis in both normal and cancerous tissue
is dependent on increased endothelial cell proliferation
and invasion, increased vessel permeability, and recruit-
ment of other support cells that make up the vessel archi-
tecture, such as pericytes. VEGF has been implicated as a
key mediator of angiogenesis in breast cancer. The most
important factor that determines survival of breast cancer
patients is dissemination of cancer cells from the primary
site into distant organs and establishment of metastatic
colonies. Comparison of gene signatures from primary
tumor, regional, and distant metastasis indicates that
VEGF is only overexpressed in distant metastasis and is
associated with poor survival.
Cyclin E is the limiting factor for G1 phase progression
and S phase entry. The cyclin E gene is a target of E2Fs,
and the protein associates with Cdk2 and activates its
kinase activity shortly before entry of cells into the S phase.
While there is evidence of the importance of cyclin D1 in
mammary tumorigenesis, the role of cyclin E in this
respect has only recently been established. Cyclin E is
expressed in supra-physiological levels in many human
cancers and its genomic locus is frequently ampliﬁed. High
levels of cyclin E and low levels of the G1-speciﬁc cell
cycle inhibitor p27KIP1 exhibit a good correlation.
Another clue for the importance of cyclin E in breast car-
cinoma is the ﬁnding of centrosome ampliﬁcations in these
tumors, which could pave the way for genomic instability.
T-box proteins contain a T-domain that affects dimeriza-
tion and DNA binding. TBX2 belongs to the Tbx subfam-
ily of T-box transcription factors. TBX2 and TBX3 are
closely related T-box proteins that have been implicated in
tissue development in different sites, including the mam-
mary gland. TBX3 is required for normal mammary
development in mouse models and in patients with ulnar–
mammary syndrome (UMS). It has been reported that
TBX2 is ampliﬁed in 8.6–21.6% of sporadic human breast
carcinomas, where the protein is overexpressed. Ectopic
expression of TBX2 results in DNA polyploidy and cispla-
tin resistance. Hence, overexpression of Tbx2 contributes
to breast carcinogenesis by accelerating cell proliferation,
changing DNA ploidy, and making cells resistant to che-
motherapy. More studies are needed to elucidate the mech-
anism of TBX2/3 overexpression in breast cancer.
TA-90 is a protein found on the outer surface of mela-
noma cells. Like S-100, TA-90 can be used to look for the
spread of melanoma. Its value in following melanoma is
still being studied, and it is not widely used at this time. It
is also being studied for use in other cancers such as colon
and breast cancer.
Cetuximab (Erbitux®) and panitumumab (Vectibix®) are
drugs targeting the EGFR protein that can be useful in the
treatment of advanced colorectal cancer. These drugs do
not work in colorectal cancers that have mutations (defects)
in the K-ras gene. Doctors now commonly test the tumor
for this gene change and only use these drugs in people
whose cancers do not have the mutation.
K-ras mutations can also help guide treatment for some
types of lung cancer. Tumors with the mutations do not
respond to treatment with erlotinib (Tarceva®) or geﬁtinib
856 The Immunoassay Handbook
CA 72-4 is a new test being studied in ovarian, pancreatic,
and stomach cancers. Studies of this marker are still in
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