TUMOR
MAKERS
Dr.
K. Harish,
M.S, D.N.B, M.Ch, F.I.C.S, F.M.S.
Associate Professor & Head
Department of Surgical Oncology
M. S. Ramaiah Medical College, Bangalore
Ever since the discovery of Bence Jones proteins in myeloma
patients (1848)1, biochemical substances have been measured
in blood and other body fluids for the purposes of managing
cancer patients.
Tumor
markers are substances that can be measured quantitatively
by biochemical or immunochemical means in tissue or body
fluids to detect a cancer and possibly the organ where
it resides, to establish the extent of tumor burden before
treatment and to monitor the response to therapy. This
procedure excludes tests like fecal occult blood and Pap
smear. Some authors include qualitative tests like immunocytostaining
of cells and certain flowcytometry studies. Earlier ‘tumor
markers’ would refer to ‘serum markers’ alone but presently
a wide range of substances are referred to as tumor markers.
Tumor
markers are classified as:
A.
Serum markers (Circulating markers)
B.
Morphologic markers & Cell surface markers
C.
Immunohistochemical markers
D.
Genetic abnormalities as biologic markers A.
SERUM
MARKERS
An
ideal marker should be highly sensitive, highly specific,
delineate the cell type of origin, provide prognostic
information, useful in treatment and indicate post treatment
progression or cure. Also, the marker must be inexpensive,
rapid to perform with reproducible results and acceptable
to patients. None of the markers available today meet
all the requirements. Given the limitations, markers have
been very useful and widely used in clinical practice.
A
range of proteins and small peptides have been identified
as secreted products of solid tumors. It is now recognized
that their production may either reflect normal genetic
expression typical of cell of origin of the cancer or
expression of genes normally suppressed.
HUMAN
CHORIONIC GONADOTROPIN (HCG)
It is generally accepted that HCG comes closest to being
an ideal tumor marker. HCG has alpha and beta subunits
and is a normal product of syncitiotrophoblast. The alpha
subunit structure is shared with the alpha subunit of
leutinising hormone (LH), follicle stimulating hormone
and thyroid stimulating hormone. The beta subunit of HCG
is unique but it still shares 80% of its amino acids with
LH. Immunoreactive HCG is identified in low concentrations
in pituitary gland, liver and colon2. There are reports
that HCG derived from the urine of choriocarcinoma patients
may differ from placental HCG through carbohydrate changes3.
Serum values are subject to less interference than urine
values.
In patients with established trophoblastic tumors following
any form of pregnancy, HCG is used to monitor the response
to therapy and to detect the development of resistance
or relapse after remission. As a rough guide, the limit
of detection of HCG (<5 IU/L) corresponds with approximately
105 cells or less than 1 mm3 of viable cells. Slow rates
of fall in HCG levels indicate a higher risk of developing
drug resistance.
Some
patients with hydatidiform mole have higher concentrations
of HCG in serum or urine than are found in corresponding
stage of pregnancy. Patients with high levels of HCG (>20000
IU/L) more than 4-5 weeks after evacuation of a hydatidiform
mole are a small subset requiring early intervention because
they are at a risk of uterine perforation. If HCG levels
fall to the limit of detection within 56 days of evacuation,
then a 6-month follow-up is generally adequate but for
others a 2-year follow-up is advisable4.
HCG
was the first demonstrated tumor marker for testis cancer5.
Germ cell tumors arising at any site may contain elements
which produce HCG whether or not typical trophoblastic
cells are identified morphologically. In these tumors,
alpha-fetoprotein is equally important and lactate dehydrogenase
may be useful. As in gestational tumors, the detection
of drug resistance and the attainment of remission and
early detection of relapses by marker measurements have
become key component in management. It has been shown
that values >10000 IU/L at diagnosis are the strongest
predictor of poor prognosis6. Serum concentrations of
beta HCG are elevated in 40-60% of patients with testicular
cancer, 80% of patients with embryonal carcinoma and 10-25%
of patients with pure seminoma. The serum half-life of
beta HCG is 24-36 hours which implies that levels should
return to normal within 5-7.5 days after surgery if all
tumor is removed.
Multiple myeloma, pancreatic carcinomas including islet
cell tumors, hepatocellular, colonic, bladder, gastric,
breast and lung cancers produce non trophoblastic immunoreactive
HCG. Serum values are usually <20ng/ml in these patients7.
ALPHA-FETOPROTEIN
(AFP):
In addition to its production in pregnancy, AFP is present
in small amounts (<10 microgram/L) in normal serum. Increased
amounts upto 107 microgram/L are found in serum of patients
with hepatocellular carcinoma and with germ cell tumors
of testes including embryonal carcinoma, yolk sac tumor,
or mixed tumors like teratocacinoma, germ cell tumors
of ovary and midline structures including mediastinum
and pineal gland. High vales of AFP, like high values
of HCG, reflecting tumor burden and perhaps aggressiveness
are of prognostic significance, so that values >500 microgram/L
indicate need for intensive therapy. Occasionally, as
a result of the action of methotrexate, platinum drugs
or etoposide on the liver, persistent elevated values
of AFP may be seen and they may not necessarily indicate
residual active tumor. Elevated AFP in a pure seminoma
or a pure choriocarcinoma indicates presence of embryonal
or yolk sac elements. The serum half-life of AFP is 5-7
days. Serum levels should return to normal within 25-35
days after all tumor has been surgically removed.
High values of AFP are also found in a large proportion
of patients with hepatocellular carcinoma. AFP of yolk
sac origin binds to concanavalin A, but there is no binding
to it by AFP of liver origin8. In Southeast Asia and Southern
Africa, AFP screening programs have been used for detecting
and confirming hepatocellular carcinomas. AFP value 100-350ng/ml
suggests and >350ng/ml usually indicates hepatocellular
carcinoma9.
AFP
could also be elevated in colonic, gastric, pancreatic
and lung cancers. Benign conditions like hepatitis, cirrhosis
and certain congenital abnormalities like ataxia telangiectasia
and neural tube defects also lead to elevated AFP levels.
PLACENTAL
ALKALINE PHOSPHATASE (PLAP):
It
is an isoenzyme of alkaline phosphatase similar to that
produced by the placenta10. Testicular seminomas are the
only tumors for which PLAP assays have consistently shown
a sensitivity >50%11, but as levels are raised only in
bulky tumors and fall to normal early in therapy it is
of marginal value. Apart from smokers, cancers of ovarian,
lung, breast and gastrointestinal origin may also result
in elevated levels.
LACTIC
ACID DEHYDROGENASE (LDH):
LDH is a cellular enzyme that catalyses the oxidation
of lactic to pyruvic acid. It has four isoenzymes that
have tissue specific distributions in normal liver, kidney
and muscle. LDH, especially isoenzyme 1 is elevated in
upto 80% of patients with all varieties of advanced testicular
germ cell tumors. The gene for isoenzyme 1 is located
on short arm of chromosome 12 (12p), which is frequently
overexpressed in the genome of germ cell tumors by formation
of an isochromosome12. Clinically LDH is a marker of bulky
disease but is less specific.
CARCINOEMBRYONIC
ANTIGEN (CEA):
Identified
in 196513, it is an oncofetal antigen normally found in
embryonic and fetal gut, pancreas and liver in the first
two trimesters of gestation. It is found on cell surface
membranes and is easily released into surrounding fluids.
According to National Institute of Health consensus, serum
levels should reach normal (<2.5ng/ml) within 6 weeks
following curative resection. Elevated levels are seen
in cigarette smokers, colorectal polyps, pancreatitis,
liver disease, pulmonary infections including tuberculosis,
alcoholic liver disease, extrahepatic biliary obstruction,
inflammatory bowel disease and renal failure. These elevations
could be due to inability to clear normal low levels of
secretion of intestinal mucosa and are usually <10ng/ml.
Elevated
levels in malignancy are non-specific and seen in colorectal
(65%), lung, pancreatic, gastric, liver, biliary tract,
thyroid, breast, uterine cervix, endometrial and ovarian
cancers.
In colonic carcinoma it is a poor marker for screening.
Poorly differentiated cancers may have normal CEA levels.
Increasing levels correlate with stage of disease. Highest
levels are seen in those with metastatic disease (liver
mets). Pre-operative raise increases the risk of recurrence.
Time to recurrence is also shorter in those with raised
levels. When pre-op CEA levels are >20ng/ml, the survival
is short for the stage. Persistent postoperative raised
values (especially raising values) predict recurrence.
Elevation occurs 2-18 months before clinical detection,
but a normal CEA does not preclude a recurrence.
Low
sensitivity and specificity makes it unsuitable for screening
purposes. It is likely that surgery, based on clinical
criteria or on a CEA blood test can improve overall survival
by only 5% for recurrent cancer14.
CA
125:
It was originally described for serous cystadenocarcinoma
of ovary15. Its half-life in the body is 20 days or more
and is the most widely employed marker for ovarian cancer.
The commonly used cut-off value is 35 U/ml.
It
is insufficiently specific and sensitive to be employed
for routine screening. It has a high negative predictive
value. The sensitivity increases to 84% in post-menopausal
women and a compromise cut-off value of 65U/ml may allow
acceptable specificity. Higher levels are seen in more
advanced and undifferentiated lesions.
Elevated
levels are seen in serous ovarian cystadenocarcinomas.
It is rarely raised in mucinous cystadenocarcinomas. Cervical
cancers, endometrial cancers, uterine sarcomas, and in
more than 40% of advanced intraabdominal neoplasms of
diverse primary site and histology. Non-malignant lesions
like cirrhosis and endometriosis may also show raised
levels.
CA
125 levels reflect response to chemotherapy or radiotherapy
after primary surgery. Though falling levels may not always
indicate a good response, a useful indicator of an objective
response is a drop in the marker level by more than 30%
after the initial course of chemotherapy and return to
a normal by 3 months. There is also a close relationship
between marker levels and later progress of disease.
PROSTATE-SPECIFIC
ANTIGEN (PSA):
It was first identified in seminal plasma16. It occurs
in normal, benign hypertrophic and cancerous prostatic
tissues but not in other normal or diseased tissues17.
PSA is detectable in serum of most men, it tends to increase
with age and rises in men with evidence of benign prostatic
hypertrophy and has a terminal half-life of 2-4 days.
Serum PSA values are altered by transurethral resection
of prostate, prostatitis, digital rectal examination and
biopsy. Using a cut-off of 10ng/ml, approximately 30%
of patients with stage I tumors and over 80% of metastatic
tumors have elevated levels, with better sensitivity than
PAP especially for soft tissue metastasis. PSA cannot
be used to predict capsular penetration. The main value
of PSA is in demonstrating persistence of disease following
surgery or radiotherapy and for monitoring the response
to hormone or chemotherapy, where it is clearly more sensitive
than PAP18. PSA levels are more sensitive in detecting
recurrent disease and may begin to rise 6-12 months before
bone scan shows recurrent disease though occasionally
progress can occur without elevation of PSA.
PROSTATIC
ACID PHOSPHATASE (PAP): It has been used extensively
for diagnosis, staging and monitoring of prostatic cancer
after its discovery19. It is ineffective for screening
of prostate cancer as it has a low positive predictive
value, low specificity and sensitivity. A raised PAP value
usually indicates an advanced disease. It is now generally
believed that PSA is superior to PAP for screening, diagnosis
and monitoring of prostatic cancers. Although levels of
PSA tend to increase in proportion to the total mass of
normal and malignant prostatic tissue, PAP levels are
much more likely to be elevated only in the presence of
metastatic disease. PAP exhibits greater specificity but
less sensitivity than PSA in detection of metastatic disease.
A brief list of some other serum markers is presented
in Table-1
TABLE
1
|
MARKER
|
TUMOR
|
| Neuron
specific enolase |
Small
cell lung cancer, neuroblastoma |
| Squamous
cell carcinomaassociated antigen |
Cervix,
other squamous tumors |
| Lipid
associated sialic acid |
Ovary,endometrium,
cervix, breast, sarcoma |
| Tissue
polypeptide antigen |
Many
advanced cancer types |
| POA
|
Pancreas |
| CA
19.9 |
Pancreas,
colorectal, ovary, upper gastrointestinal tract |
| CA
50 |
As
CA 19.9, breast, lung, prostate |
| CA
72 |
Colorectal,
pancreas, ovary |
| CA
15.3 |
Breast,
ovarian, genital tract cancers |
| TAG
72.3 |
Ovarian,
colon |
| DU
PAN 2 |
Pancreas,
other upper gastrointestinal tract |
| NB/70K |
Ovary,
breast, lung, gastrointestinal tract |
| Tumor
associated trypsin inhibitor |
Ovary,
pancreas |
| Polymorphic
epithelial mucin |
Breast,
ovary |
| ACTH |
Lung,
ovarian, carcinoid, thymoma, islet cell tumors, thyroid
medullary carcinoma |
| ADH
|
Lung,
carcinoid |
| Throglobulin |
Well-differentiated
thyroid cancer |
| Calcitonin
|
Medullary
carcinoma thyroid |
| 5-HIAA |
Carcinoid |
| PTH
|
Lung,
renal, hepatoma, breast |
| Gastrin,
insulin, glucagon, VIP |
Corresponding
pancreatic islet tumor |
| Immunoglobulins
(M component) |
Myeloma,
macroglobinemia, lymphoma |
| Ferritin |
Hodgkin’s
lymphoma, lung, pancreas, ovary |
| Hydroxy-proline |
Multiple
Myeloma, breast, pancreas |
| CYFRA
21-1 |
Non-small
cell lung cancer |
B.
MORPHOLOGIC & CELL SURFACE MARKERS
The
development of cell surface modifications and the character
of cytoplasmic organelles form the basis for evaluating
cytoplasmic differentiation. Better-differentiated tumors
have better developed ultrastructural features. Organellar
features associated with specific tumors are summarized
in Table-2.
TABLE
2
| Cell
junctions |
Typical
of carcinomas but can be found in other tumor types |
| Tonofilament/Desmosomalcomplexes |
Squamous
cell carcinomas |
| Basal
lamina |
Well
developed carcinomas, schwann and smooth muscle tumors
|
| Microvilli |
Typical
of adenocarcinomas, very long in mesotheliomas, hairy
cell leukemia |
| Cilia |
Carcinomas
esp. lung, female genital tract, some mesenchymal
tumors |
| Intracytoplasmic
lumens |
Adenocarcinomas,
some mesotheliomas |
| Pinocytic
vesicles |
Smooth
muscle and endothelial tumors |
| Mitochondria |
Abundant
in oncocytomas |
| Smooth
endoplasmic reticulum |
Steroid
producing cells |
| Annulate
lamellae |
Typical
of dysgerminomas (seminomas) |
| Lysosomes
|
Granular
cell and histiocytic tumors |
| Teleolysosomes |
Urothelial
and Brenner’s tumors |
| Langerhan’s
(Birbek) granules |
Histiocytic
tumors |
| Mucin
granules |
Mucin
producing adenocarcinomas |
| Bile |
Hepatomas |
| Neuroendocrine
granules |
Endocrine
/ neural tumors |
| Melanosomes |
Melanomas,
pigmented neurofibromas |
| Dense
plaques |
Smooth
muscle tumors, fibrous histiocytomas |
| Z
bands |
Skeletal
muscle tumors |
C.
IMMUNOHISTOCHEMICAL MARKERS
It
refers to a broad group of methodologies, which take advantage
of the ability of specific antibodies to identify unique
antigenic determinants, in cells and tissues. These may
include structural elements, enzymes, hormones, receptors,
‘tumor-specific’ antigens, etc. In general, tissue antigens
are detected through a chain of antigen-antibody reactions
using the tissue section as the site of initial antigen.
A primary antibody specific for the marker under study
is applied and will bind to the tissue section if antigenic
sites are recognized in specific antibody-antigen reactions.
This reaction is amplified using sequential antibodies
directed against the previously applied immunoglobulin
preparation. Visual detection of these reactions is accomplished
by labeling one of the secondary antibodies with an enzyme,
usually Hydrogen Peroxidase, and developing the reaction
with specific substrate, in this case hydrogen peroxidase,
and a chromagen such as diaminobenzidine. Brown granules
visualize a positive reaction. Cytoplasmic immunoglobulins
are well preserved with formalin and paraffin sections,
but intermediate filaments and plasma membrane immunoglobulins
are better seen with frozen section.
Clinical
applications:
1.
Providing broad subclassification of poorly differentiated
or morphologically non-classifiable tumors. For instance,
spindle cell neoplasms of skin may represent poorly differentiated
Squamous cell carcinomas, melanoma, fibrohistiocytic or
neural tumors. Similarly, ‘small blue cell tumor’ includes
diverse lesions like lymphoma, neuroblastoma, rhabdomyosarcoma,
Ewing’s sarcoma and small cell carcinoma.
2.
Precisely identifying the site of origin of metastatic
lesions. In this setting, the histologic diagnosis (like
adenocarcinoma) may be straightforward, but the clinical
evaluation does not disclose the primary site. Various
monoclonal antibodies are used to identify the specific
cellular products. These antibodies will not distinguish
between neoplastic and non-neoplastic states but can suggest
the organ of origin. Examples of tumor specific antigens
include PSA (for prostate), throglobulin (for thyroid),
lactalbumin (for breast), myoglobin (for muscle cells),
salivary gland amylase (for salivary gland) and pancreatic
amylase (for pancreas).
3.
Establishing similarities between primary and metastatic
or recurrent lesions.
4.
Confirming diagnosis made by histologic criteria.
5.
Distinction of malignant lesions from morphologically
similar benign or non-neoplastic conditions. For example
squamous cell carcinoma of cervix and cervicitis. The
search continues, though in practice few, if any have
been identified. Antigens expressed in human malignancies:
1. Intermediate filaments: constitute a class of cytoskeletal
cellular components present both in normal and neoplastic
cell. They are outlined in Table 3.
TABLE
3
|
CLASS
|
NAME
|
DISTRIBUTION
|
| I
|
Acidic
cytokeratins |
Epithelial
cells |
| II
|
Neutral,
basic cytokeratins |
Epithelial
cells |
| III |
Vimentin,
Glial fibrillary acid protein Desmin |
Mesenchymal
cellsGlial cells |
| IV |
Neurofilament |
Neurons |
| V |
Lamins
A and C |
Nuclei |
2.
Other markers of cell lineage: Epithelial membrane antigen
(EMA) is expressed on the surface of most epithelial cells
except hepatocellular and adrenal cortical carcinomas.
Leukocyte common antigen (LCA) usually distinguishes lymphomas
from other malignancies. Melanocytes, glial cells, Schwann
cells, neurons, salivary glands, chondrocytes and Langerhans
cells express S-100 protein. Factor VIII related angiosarcomas
and Kaposi’s sarcoma express antigen.
3.
Oncofetal antigens: CEA, AFP and HCG are widely studied
and used.
4.
Tumor specific products in adenocarcinoma: same as No.
2 outlined under ‘clinical applications’ above.
D.
GENETIC MARKERS
It
includes chromosomal markers and oncogenes. The chromosomal
markers are summarized in Table-4.
TABLE
4
|
|
MARKER
|
EXAMPLE
|
| DIAGNOSIS |
Tumor
specific translocation |
t(9;22),
CML |
| Tumor
specific monosomy |
-22,
meningioma |
| Clonal
aberrations |
+8,
ANLL |
| PREDISPOSITION |
Increased
chromosome breakage |
Radiation |
| Constitutional
chromosome aberration |
Leukemia
in trisomy 21 |
|
Aberration
superimposed on original clonal marker |
t(9;22), +8; blast CML |
| PROGRESSION |
Multiclonality |
Glioblastoma |
|
Gene
amplification |
Double
minute or HSR regions in neuroblastoma |
Some of the characteristic karyotypic abnormalities in
solid tumors are shown in Table-5.
TABLE
5
|
Tumor
type |
Type
of rearrangement |
| Epithelial
tumors |
Pleomorphic
adenoma |
t(3;8)(p21;q12) |
| Lung
carcinoma |
del(3)(p14-23) |
| Wilms
tumor |
del(11)(p13) |
| Mesenchymal
tumors |
Synovial
sarcoma |
t(X;18)(p11;q11) |
| Rhabdomyosarcoma |
t(2;13)(q35-37;q14) |
| Liposarcoma(myxoid) |
t(12;16)(q13.3;p11.2)
|
|
Neuroblastoma
|
del(1)(p32;36) |
| Neurogenic
and others |
Retinoblastoma |
del(13)(q14) |
| Ewings
sarcoma |
t(11;22)(q24;q12) |
| Germ
cell tumors |
i(12p) |
Genetic tumor markers are summarized in Table-6.
TABLE
6
| Type
of genetic change |
Gene
|
Tumor |
|
Gene
amplification
Gene
mutation
Loss
of tumor suppressor gene
|
c-erb
B-2 |
Breast,
ovary, stomach |
| N-myc |
Neuroblastoma |
| c-myc
|
Lung |
| as |
Pancreas,lung,
colorectal,kidney |
| APC
|
Colorectal
|
| Rb-1 |
Retinoblastoma,
breast |
|
p53 |
Lung,
breast, colorectal |
Summary
of clinical application of cancer markers:
Diagnosis:
Hormone
markers of endocrine tumors (HIAA)
Serum concentration (monoclonal immunoglobulin, CEA, AFP)
Cell surface markers Tissue
localization (CEA, others)
Radioimmune scintigraphy (CEA, AFP)
Prognosis:
Serial
determination (CEA, AFP, HCG etc.)
Follow-up
for recurrent disease:
Therapy
(experimental):
Antibody
(CEA, T cell)
Antibody-drug conjugate (Ricin)
Radiolabelled antibody
Radioimmunoguided surgery
The
clinical applications of tumor markers have not fulfilled
all the optimistic expectations in diagnosis and management.
Nevertheless, markers have had tremendous impact in management
of cancers. Search for new markers is more than justified
by the vast potential for clinical applications. Newer
areas of therapy like radiolabelled antibody imaging and
radioimmunoguided surgery are being explored. Overall
markers have added a new dimension to managing cancers.
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SUGGESTED
FURTHER READING:
(a)
Oxford Textbook of Oncology, Oxford Medical Publications,
Oxford University Press, 1st Ed., 1995.
(b)
Cancer, Principles and Practice of Oncology, J B Lippincott
Company, 5th Ed., 1997.
(c)
Comprehensive Textbook of Oncology, Williams and Wilkins,
2nd Ed., 1991.
(d)
Campbell’s Urology, W B Saunders Company, 7th Ed., 1998.
(e)
Clinical Oncology, Churchill Livingstone, 1st Ed., 1995.
(f)
Gynecologic Oncology, Fundamental principles and Clinical
Practice, Churchill Livingstone, 2nd Ed., 1992.
(g)
Textbook of Surgery, The biological basis of modern surgical
practice, 15th ed., 1997.
(h)
The Urologic Clinics of North America, Testis Cancer,
Vol. 25, No. 3, Aug 1998.
(i)
The Urologic Clinics of North America, Testis Cancer in
adults and children, Vol. 20, No. 1, Feb 1993.
(j) The Urologic Clinics of North America, Prostatic tumor
markers, Vol. 20, No. 4, Nov 1993.
(k)
The Surgical Clinics of North America, Colorectal Cancer,
Vol. 73, No. 1, Feb 1993.
