Circulating Tumor Markers: Concepts & Clinical Applications

1. Chapter 32: Circulating Tumor Markers: Basic Concepts and Clinical Applications By Christopher R. McCudden, Monte S. Willis

2. Introduction • Cancer • 2nd leading cause of death in developed countries • Accounts for more than 2.7 million deaths annually • Is uncontrolled growth of cells that can develop into a tumor & spread to other areas of body • Formation & spreading (metastasis) of tumors is caused by a complex combination of inherited & acquired genetic mutations. • In formation, mutations include activation of growth factors & oncogenes & inhibition of apoptosis, tumor suppressor, & cell cycle regulation genes. • Cancer is staged based on tumor size, histology, regional lymph node involvement, & presence of metastasis; 4 stages (I–IV).

3. Introduction (cont’d) • Cancer staging and progression

4. Types of Tumor Markers • Function and Origin • Detect & monitor cancer • Produced by tumor directly or as an effect of tumor on tissue • Types • Enzymes: levels of certain enzymes correlate with tumor burden • Serum proteins:2-microglobulin & immunoglobulins • Hormones & metabolites: specific markers of secreting tumors • Oncofetal antigens: carcinoembryonic, alpha-fetoprotein • Receptors: non-serologic; estrogen & progesterone receptors

5. Types of Tumor Markers (cont’d) • Timeline of tumor marker use

6. 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).

7. 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.

8. Laboratory Considerations for Tumor Marker Measurement • Multiple tests should be performed, using same commercial kits. • Lack of standardization • Serially evaluate tumor markers, because they increase with time, whereas high normal values will not.

9. 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

10. 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

11. 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

12. 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.

13. 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)

14. 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

15. 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

16. 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

17. 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 kDa • Is involved in apoptosis, immunity, & cell adhesion

18. 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.

19. 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

20. 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

21. 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