TUMOR MARKERS

CA-125

• CA-125 is an antigen present on 80% of non-mucinous ovarian

carcinomas. It is defined by a monoclonal antibody that was
generated by immunizing laboratory mice with a cell line
established from human ovarian carcinoma. It circulates in the
serum of patients with ovarian carcinoma and was therefore
investigated for possible use as a marker.
• CA-125 is often elevated in patients with ovarian cancer, its level following the patient’s
clinical course. With surgical resection or chemotherapy, the level correlates with patient
response. Thus, it is superior to other markers such as CEA.
• The CA-125 is elevated in other cancers including endometrial, pancreatic, lung, breast,
and colon cancer, and in menstruation, pregnancy, endometriosis, and other gynecologic
and non-gynecologic conditions.
 PRINCIPLE OF THE TEST

• The CA-125 ELISA test is based on the principle of a solid phase enzyme-linked immunosorbent
assay. The assay system utilizes monoclonal antibody directed against a distinct antigenic
determinant on the intact CA-125 molecule is used for solid phase immobilization (on the
microtiter wells). A rabbit anti-CA-125 antibody conjugated to horseradish peroxidase is in the
antibody-enzyme conjugate solution. The test sample is allowed to react simultaneously with the
two antibodies, resulting in the CA-125 molecules being sandwiched between the solid phase and
enzyme-linked antibodies. After incubation at 37C for 90 minutes, the wells are washed with water
to remove unbound-labeled antibodies. A solution of TMB Reagent is added and incubated for 20
minutes, resulting in the development of a blue color. The color development is stopped with the
addition of Stop Solution changing the color to yellow. The concentration of CA-125 is directly
proportional to the color intensity of the test sample. Absorbance is measured
spectrophotometrically at 450nm.
 ASSAY PROCEDURE

1. Secure the desired number of coated wells in the holder.

2. Dispense 100ul of CA-125 standard, specimens, and controls into appropriate wells.
3. Dispense 100ul of Enzyme conjugate reagent to each well.
4. Thoroughly mix for 30 seconds. It is very important to have a complete mixing in the setup.
5. Incubate at 37C for 90 minutes.
6. Remove the incubation mixture by emptying plate content into a waste container.
7. Rinse and empty the microtiter plate 5 times with distilled or deionized water.
8. Strike the microtiter plate sharply onto absorbent paper or paper towels to remove all residual water droplets.
9. Dispense 100ul of TMB Reagent into each well. Gently mix for 10 seconds.
10. Incubate at room temperature. In the dark, for 20 minutes.
11. Stop the reaction by adding 100ul of Stop Solution to each well.
12. Gently mix for 30 seconds. It is important to make sure that all the blue color changes to yellow color completely.
13. Read the optical density at 450nm with a microtiter plate reader within 15 minutes.
 EXPECTED VALUES AND SENSITIVITY

• Healthy women are expected to have CA-125 assay values below 35U/ml. the minimum
detectable concentration of CA-125 in this assay is estimated to be 5U/ml.s
ENZYME TUMOR TUMOR TYPE METHOD SPECIMEN CLINICAL UTILITY
MARKERS
Prostate-specific Prostate cancer IA Serum Prostate cancer
antigen screening, therapy
monitoring and
recurrence
Lactate dehydrogenase Hematologic EA Serum Prognostic indicator;
malignancies elevated
nonspecifically in
numerous cancers
Alkaline phosphatase Metastatic carcinoma EA Serum Determination of liver
of bone, hepatocellular and bone involvement;
carcinoma, nonspecific elevation
osteosarcoma, in many bone-related
lymphoma, leukemia and liver cancers
Neuron-specific Neuroendocrine RIA, IHC Serum Prognostic indicator
enolase tumors and monitoring disease
progression for
neuroendocrine tumor
 SERUM PROTEIN TUMOR TYPE METHOD SPECIMEN CLINICAL
TUMOR MARKERS UTILITY

Serum M protein Plasma cell dyscrasias SPE/IFE Serum Diagnosis, therapeutic

monitoring of plasma
cell malignancies

Serum free light Plasma cell dyscrasias IS Serum Diagnosis, therapeutic

chains monitoring of plasma
cell malignancies

B2-macroglobulin Hematologic IA Serum Prognostic marker for

malignancies lymphoproliferative
disorders
 ENDOCRINE TUMOR TYPE METHOD SPECIMEN CLINICAL UTILITY
TUMOR MARKERS

Homovanillic acid Neuroblastoma, HPLC 24-h urine Diagnosis of

(HVA) Pheochromocytoma, neuroblastoma
Paraganglionoma

Vanillylmandelic acid Pheochromocytoma, HPLC 24-h urine Diagnosis of

Paraganglionoma, neuroblastoma
Neuroblastoma

Metanephrines Pheochromocytoma, HPLC 24-h urine Screening and

Paraganglionoma, Or plasma diagnosis of
Neuroblastoma pheochromocytoma

Catecholamines Pheochromocytoma, HPLC; LC-MS/MS 24-hr urine Screening and

Paraganglionoma, diagnosis of
Neuroblastoma pheochromocytoma,
paraganglionoma,
neuroblastoma
 ENDOCRINE TUMOR TYPE METHOD SPECIMEN CLINICAL
TUMOR MARKERS UTILITY

Hydroxyindoleacetic Carcinoid tumors HPLC 24-hr urine Diagnosis of carcinoid

acid (5-HIAA) tumors

Serotonin Carcinoid tumors HPLC Serum Diagnosis of carcinoid

tumors

Calcitonin MTC and IA Serum Screening, response to

neuroendocrine tumors therapy, and
monitoring recurrence
of MTC
Parathyroid hormone Pituitary adenoma IA Serum Diagnosis and
postsurgical
monitoring of
hyperparathyroidism
 ENDOCRINE TUMOR TYPE METHOD SPECIMEN CLINICAL
TUMOR UTILITY
MARKERS

Growth hormone Pituitary adenoma, IA Serum Diagnosis and post

ectopic GH-secreting monitoring of
tumorsurgical acromegaly

Prolactin Pituitary adenoma IA Serum Diagnosis and

postsurgical
monitoring of
prolactinoma
Adrenocorticotropic Pituitary adenoma, IA Serum Diagnosis of ectopic
hormone ectopic ACTH- ACTH-producing
producing tumor tumor

Cortisol Adrenal tumors IA Serum or urine Diagnosis of

Cushing’s syndrome
 ENDOCRINE TUMOR TYPE METHOD SPECIMEN CLINICAL
TUMOR UTILITY
MARKERS

Antidiuretic hormone Posterior pituitary IA Serum Diagnosis of SIADH

tumors

Chromogranin A Pheochromocytoma, ELISA, RIA Serum Aid in diagnosis of

Neuroblastoma, carcinoid tumors,
carcinoid tumors, pheochromocytomas,
small cell lung and neuroblastomas
cancers
C-peptide Insulin-secreting ELISA, RIA Serum Diagnosis of
tumors insulinoma
CARBOHYDRATE TUMOR TYPE METHOD SPECIMEN CLINICAL UTILITY
AND CANCER
ANTIGEN TUMOR
MARKERS

CA 19-9 GI cancer and Immunoassay Serum Monitoring

adenocarcinoma pancreatic cancer

CA 15-3 Metastatic breast Immunoassay Serum Response to therapy

cancer and detecting
recurrence

CA 27-29 Metastatic breast Immunoassay Serum Response to therapy

carcinoma and detecting
recurrence

CA-125 Ovarian cancer Immunoassay Serum Monitoring therapy

 RECEPTOR TUMOR TYPE METHOD SPECIMEN CLINICAL
TUMOR UTILITY
MARKERS

Estrogen receptor Breast cancer IHC Biopsy Hormonal therapy

indicator

Progesterone Breast cancer IHC Biopsy Hormonal therapy

receptor indicator

Her-2/neu Breast, ovarian, IHC, FISH, Biopsy Prognostic and

GIT tumors ELISA hormonal therapy
indicator

Epidermal growth Head, neck, IHC Biopsy Prognostic

factor receptor ovarian, cervical indicator
cancers
 IMMUNOASSAYS

• The most commonly used method to measure tumor markers.

• Ability to automate testing
 HIGH-PERFORMANCE LIQUID
CHROMATOGRAPHY
• The most widely used methodology to detect catecholamines and their metabolites in
plasma and urine.
 IMMUNOHISTOCHEMISTRY

• Performed by thinly cutting and placing the tissue in question on glass slides. Specific
antibodies in solution are then incubated with tissue sections to the detect the presence of
antigens using colorimetric secondary antibodies.
END OF DISCUSSION