Tumor Markers

• Cancer – is the second leading cause of death in North

America, accounting for more than 500,000 deaths annually.

– It is estimated that 45% of males and 38% of females will develop invasive cancer in their lifetime.

• Cancer – refers to the uncontrolled growth of cells that can

develop into a solid mass or tumor and spread to other areas of the body.

– Are caused by a complex combination of inherited and acquired genetic mutations

Tumorigenesis• Formation

• activation of:– growth factors (e.g., epidermal growth factor [EGF])– oncogenes (e.g., K-ras)

• encodes a protein that, when mutated, promotes uncontrolled cell growth

• inhibition of:– Apoptosis

• programmed cell death

– tumor suppressor• encodes a protein involved in protecting cells from unregulated growth

– cell cycle regulation genes (e.g., BRCA1, p53, cyclins).

Metastasis

– spreading

– loss of cell adhesion proteins (e.g., -catenin and E-cadherin)

– activation of angiogenesis genes • Angiogenesis:

– development of new blood vessels to supply oxygen and nutrients to cells

• Cancer severity is generally classified by a combination of several factors: – tumor size– histology– regional lymph node involvement– presence of metastasis

• For most solid tumors (e.g., breast, lung, kidney), cancer is broadlyclassified into four stages:• Stage I• Stage II• Stage III• StageIV

Cancer Staging and Progression

Tumor markers• are produced either directly by the tumor or as an effect of the tumor on healthy tissue (host).

• Types:1. serum proteins

• also used to monitor cancer therapy– 2-microglobulin

» found on the surface of all nucleated cells and can therefore be used as a nonspecific marker of the high cell turnover that is often observed in tumors

– immunoglobulins» provide a more specific measure of plasma cell production of monoclonal proteins observedin hematologic malignancies

such as multiple myeloma

2. Oncofetal antigens • carcinoembryonic, alpha-fetoprotein

3. Hormones & Metabolites• specific markers of secreting tumors• particular valuable in diagnosing neuroblastomas, as well as pituitary and adrenal adenomas

4. Receptors• non-serologic; estrogen & progesterone receptors• used to classify tumors for therapy

5. Enzymes • levels of certain enzymes correlate with tumor burden

6. carbohydrate or cancer antigens• are best used for monitoring treatment of tumor that secrete these epitopes

• Timeline of tumor marker use

Applications of Tumor Marker Detection

• Screening– Most tumor markers are found in normal cells, not just cancer cells.– Therefore, screening asymptomatic populations would result in detection of

false positives, causing undue alarm & cost.– Few tumor markers are used to screen populations.– Susceptibility to breast, ovarian, & colon cancer can be determined by

identifying germline mutations in patients with a family history of these diseases.

– Breast & ovarian cancers are associated with germline ---BRCA1 & BRCA2 mutations,

– colon cancer with ----adenomatous polyposis coli gene (APC).

Applications of Tumor Marker Detection (cont’d)• Prognosis

– Tumor marker concentration gradually increases with tumor progression, reaching highest levels when tumors metastasize.

– Tumor marker levels at diagnosis can reflect presence of malignancy & aggressiveness of tumor & help predict outcome.

• Monitoring Therapy Effectiveness & Disease Recurrence– After surgical resection, radiation, or chemotherapy, tumor markers are

observed.– Effective therapy can result in decrease in tumor markers.– Appearance of tumor markers after effective therapy can be used as a highly

sensitive marker of recurrence.

Recommendations for Test Ordering

• Multiple tests should be performed, using same commercial kits.

• Follow-up testing should be based on half-life of marker to ensure that pre-existing marker has had time to clear.

• Serially evaluate tumor markers, because they increase with time, whereas high normal values will not.

Methods

• Standardization found for other common clinical assays generally does not exist for cancer assays.

• Comparisons of results from different assays for a single patient can be treacherous due to differences in:– Antibody specificity– Analyte heterogeneity– Assay design– Lack of standard reference material– Calibration & kinetics– Variation in reference ranges

Methods (cont’d)

• Immunoassays– Most commonly used method to measure tumor markers– Have many advantages, including ability to automate testing– Factors in interpreting tumor marker immunoassays: linearity, hook effect, &

heterophile antibodies– Linearity

• Linear range: range of analyte concentrations in which a linear relationship exists between analyte & signal

• Measured by analyzing specimens spanning reportable range

Methods (cont’d)

• Immunoassays– Hook effect

• Falsely low measurements as a result of excessively high tumor marker concentrations

• Capture & label antibodies are saturated, resulting in a lack of a “sandwich” formation, resulting in decrease in signal.

• Samples exceeding linear range should be diluted & retested.– Heterophile antibodies

• Circulating antibodies against animal immunoglobulins can cause significant interference in immunoassays.

• Occur in patients given mouse monoclonal antibodies

Methods (cont’d)

• High-Performance Liquid Chromatography (HPLC)– Most widely used method to detect catecholamines & their metabolites in

plasma & urine– Analytes of interest are separated from plasma or urine, run over a column, &

separated by physical characteristics.– Used to detect neuroblastoma, pheochromocytoma, carcinoid tumors

• Immunohistochemistry (IHC)– Tests tumor markers that are detected directly within solid tissue– Slice of tissue is placed on glass slide & incubated with specific antibodies in

solution to detect presence of antigens.

Immunohistochemistry (IHC)(cont’d)

• A good example of the use of a tumor marker that is detected by immunohistochemistry (IHC) is the identification of estrogen and progesterone receptors in breast cancer.

• When breast tumors are positive for estrogen and progesterone receptors at the cell surface, they tend to respond to hormonal therapy.

• While tumors lacking these receptors are treated with other chemotherapeutic modalities.

Methods (cont’d)

• Enzyme Assays– Detection of elevated circulating enzymes generally cannot be used to identify

a specific tumor or site of tumor.– Exception: prostate specific antigen (PSA), found exclusively in diseased &

benign prostate glands– Before use of immunoassays & oncofetal antigens was common, enzyme

detection was widely used.– Examples of enzymes used as tumor markers: alkaline phosphatase (bone,

liver, leukemia, sarcoma), creatine kinase-BB (prostate, small-cell lung, breast, colon, ovarian), lactate dehydrogenase (liver, lymphomas, leukemia), & PSA (prostate)

Frequently Ordered Tumor Markers

• Alpha-Fetoprotein (AFP)– Introduction/Description

• An abundant serum protein synthesized by fetal liver & re-expressed in certain types of tumors

• Often elevated in patients with hepatocellular carcinoma & germ cell tumors

– Regulation/Physiology• A 70-kd glycoprotein related to albumin; normally functions as a transport

protein & is involved in regulating oncotic pressure in fetus• Upper limit of normal for serum AFP: ~15 ng/mL in adults

Frequently Ordered Tumor Markers (cont’d)

• Alpha-Fetoprotein (AFP)– Clinical Application/Interpretation

• Used for diagnosis, staging, prognosis, & treatment monitoring of hepatocellular carcinoma

• Also used for classification & monitoring therapy for testicular cancer & for tumor staging

– Methodology• Measured by a variety of immunoassays

Frequently Ordered Tumor Markers (cont’d)

• Cancer Antigen 125 (CA-125)– Introduction/Description

• May be useful for detecting ovarian tumors at an early stage & for monitoring treatments without surgical restaging

– Regulation/Physiology• Expressed in ovary, other tissues of müllerian duct origin, & ovarian

carcinoma cells– Clinical Application/Interpretation

• Only clinically accepted serologic marker of ovarian cancer– Methodology

• Immunoassays using “OC 125” & “M11” antibodies

Frequently Ordered Tumor Markers (cont’d)

• Carcinoembryonic Antigen (CEA)– Introduction/Description

• Discovered in 1960s; prototypical example of oncofetal antigen• Expressed during development & re-expressed in tumors• Most widely used tumor marker for colorectal cancer; also elevated in

lung, breast, & GI tumors– Regulation/Physiology

• Large heterogenous glycoprotein; molecular weight: ~200 kd• Is involved in apoptosis, immunity, & cell adhesion

Frequently Ordered Tumor Markers (cont’d)

• Carcinoembryonic Antigen (CEA)– Clinical Application/Interpretation

• A tumor marker for colorectal cancer• Used for prognosis, post-surgery surveillance, & to monitor response to

chemotherapy– Methodology

• Historically used polyclonal antibodies; now uses monoclonal anti-CEA antibodies

• Due to high heterogeneity of CEA, it is essential that same assay be used for serial monitoring.

Frequently Ordered Tumor Markers (cont’d)

• Human Chorionic Gonadotropin (hCG)– Introduction/Description

• A dimeric hormone secreted by trophoblasts in placenta to maintain corpus luteum during pregnancy

– Regulation/Physiology• A 45-kd glycoprotein consisting of alpha & beta subunits

– Clinical Application/Interpretation• Prognosis of ovarian cancer, diagnosis of testicular cancer, most useful

marker for gestational trophoblastic diseases– Methodology

• Immunoassays w/ monoclonal capture, tracer antibodies

Frequently Ordered Tumor Markers (cont’d)

• Prostate Specific Antigen (PSA)– Introduction/Description

• A 28-kd glycoprotein produced only in epithelial cells of acini & in prostatic ducts

• Regulates seminal fluid viscosity• Dissolves cervical mucous cap, allowing sperm to enter

– Regulation/Physiology• Low levels of PSA can be detected in serum of healthy men.• Two major forms found circulating in blood: free & complexed

Frequently Ordered Tumor Markers (cont’d)

• Prostate Specific Antigen (PSA)– Clinical Application/Interpretation

• Annual screening of prostate cancer in men over 50 years old & in younger men at high risk (family history)

• Normal range for PSA: <4 ng/mL• Factors to take into account when testing for PSA: age, PSA velocity, free

PSA/total PSA ratios– Methodology

• Measured by immunoassay using enzyme, fluorescence, or chemiluminescence on an automated immunoassay platform