In tumor immunology, a fundamental tenet is that when a nor mal cell is transformed into a malignant cell, it develops unique antigens not normally present on the mature normal cell.
Tumors frequently produce tumor-specific antigens (TSAs) to which the host may develop antibodies. Virus-induced cancers are the most antigenic; chemical-induced cancers are the least antigenic.
Tumor markers are substances present in or produced by tumors that can be used to detect the presence of cancer based on their measurement in blood, body fluids, cells, or tissue (Table 1). A tumor marker may be produced by the host in response to a tumor that can be used to differentiate a tumor from normal tissue or to determine the presence of a tumor. Non-neoplastic conditions can also exhibit tumor marker activity (Table 2). Some tumor markers are used to screen for cancer, but markers are more often used to monitor recurrence of cancer or determine the degree of tumor burden in the patient. To be of any practical use, the tumor marker must be able to reveal the presence of the tumor while it is still susceptible to destructive treatment by surgical or other means. Tumor markers can be measured quantitatively in tissues and body fluids using biochemical, immunochemical, or molecular tests (Table 3).
Table1. Cancer Biomarkers
Table2. Non-neoplastic Conditions With Elevated Serum and Plasma Concentrations of Tumor Markers
Table3. Tumor Markers in Neoplasms
The search for tumor markers goes back more than 150 years. The earliest identified tumor marker was Bence-Jones protein, a light-chain immunoglobulin, found in patients with multiple myeloma. Over the last 15 years, the use of tumor markers in the United States has risen dramatically. Tumor markers play an especially important role in the diagnosis and monitoring of patients with prostate, breast, and bladder cancers.
Older, well-established markers include alkaline phosphatase and collagen-type markers in bone cancer, immunoglobulins in myeloma, catecholamines and their derivatives in neuroblastoma and pheochromocytoma, and serotonin metabolites in carcinoid. In addition, there are many breast tissue prognostic markers (e.g., hormone receptors, cathepsin-D, HER2/neu oncogenes, plasminogen receptors and inhibitors).
The list of tumor markers approved by the U.S. Food and Drug Administration (FDA) continues to grow (Table 4). Nine of these biomarkers are protein biomarkers identifiable in blood. Other recently approved protein biomarkers can be detected in urine, such as nuclear matrix protein 22, fibrin and fibrinogen degradation products, and bladder tumor antigen for monitoring bladder cancer, and by immunohistochemical methods using tumor tissues, such as estrogen receptor for breast cancer. Additional FDA-approved cancer biomarkers are DNA based, such as human epidermal growth factor receptor 2 and HER2/neu for breast cancer, and can be assayed by fluorescent in situ hybridization (FISH). Multiple-marker combinations are useful in the management of some cancers (Table 5), but the use of more than two markers is questionable.
Table4. Some Common Serum Tumour Markers and Their Clinical Utility*
Table5. Related Multiple Tumor Markers
An ideal tumor marker would be an assay in which a positive result would only occur in patients with a malignancy, would correlate with stage and response to treatment, and is easily reproducible. No tumor marker to date has met this ideal marker description, nor has any tumor marker has been established as a practical screening test in a general healthy population or in most high-risk populations. The rationale for this poor predictive value of tumor markers is the lack of sensitivity and specificity in the low cancer rates that prevail in population groups. Because of the low prevalence of cancer, in general, even assays that are highly sensitive and specific may have a low predictive value.
Categories of Tumor Antigens
Tumor cells manifest tumor antigens, as well as self HLA anti gens. There are four types of tumor antigens identified:
1. Tumor-specific antigens (TSAs) on chemically induced tumors
2. Tumor-associated antigens (TAAs) on virally induced tumors
3. Carcinofetal antigens
4. Spontaneous tumor antigens
Tumor-Specific Antigens
Chemically induced tumors are known to develop TSAs, which are uniquely associated with each tumor. These antigens are not found in normal cells. TSAs demonstrate little or no cross reactivity between different tumors caused by the same carcinogen, perhaps because every tumor caused by chemical agents has unique surface characteristics.
Tumor-Associated Antigens
TAAs are cell surface molecules coded for by tumorigenic viruses. These antigens are not expressed on the virion but are synthesized by the host cell. In contrast to TSAs, TAAs are virus-specific. Therefore, each specific virus induces the same antigens, regardless of the tissue of origin or the animal species.
Carcinofetal Antigens
Well-differentiated tissue produces and secretes little or no fetal gene products. The abnormal behavior of malignant cells is believed to derepress genes normally expressed only during fetal life. Because the products of these fetally active genes are recognized as self, they do not elicit humoral or cell-mediated responses.
During malignant transformation, however, gene derepression is responsible for the production of increased concentrations of these gene products, which are known as oncofetal proteins. Carcinoembryonic antigen is an example of a carcinofetal antigen.
Spontaneous Tumor Antigens
Tumors caused by no known mechanism, known as spontaneous tumor antigens, are thought to produce antigens. Disagreement exists regarding whether these tumors are similar to those produced experimentally by chemical, viral, or physical agents. Although substantial evidence supports the contention that these tumors do not produce unique antigens, some evidence has refuted this contention. The importance of these findings remains unclear.
Specific Tumor Markers
Ten protein cancer biomarkers have been FDA-approved for clinical use:
1. Alpha-fetoprotein
2. CA 125
3. Human epididymis protein 4
4. Thyroglobulin
5. Prostate-specific antigen (PSA)
6. Carcinoembryonic antigen
7. CA 19-9
8. CA 15-3
9. CA 27.29
10. HER2/neu
Other markers include the beta subunit of human chorionic gonadotropin (β-hCG) and miscellaneous enzyme and hormone markers.
Alpha-Fetoprotein
Alpha-fetoprotein (AFP) is normally synthesized by the fetal liver and yolk sac. AFP is secreted in the serum in nano gram to milligram quantities in hepatocarcinoma, endodermal sinus tumors, nonseminomatous germ cell (testicular) cancer, teratocarcinoma of the testis or ovary, and malignant tumors of the mediastinum and sacrococcyx. In addition, a small percentage of patients with gastric and pancreatic cancer with liver metastasis may have elevated AFP levels. Both AFP and β-hCG should be quantitated initially in all patients with teratocarcinoma because one or both markers may be secreted in 85% of patients. The concentration of AFP may be elevated in nonneoplastic conditions such as hepatitis and cystic fibrosis.
AFP is a reliable marker for following a patient’s response to chemotherapy and radiation therapy. Levels should be obtained every 2 to 4 weeks (metabolic half-life in vivo, 4 days).
CA 125
CA 125, a mucin-like glycoprotein, is expressed on the surface of coelomic epithelium and human ovarian carcinoma cells. CA 125 is relatively more sensitive in low-stage ovarian cancer. It reacts against an MAb developed against a cell line from one patient’s ovarian cystadenocarcinoma. It is elevated in carcinomas and benign disease of various organs (e.g., pelvic inflammatory disease, endometriosis), but is most useful in ovarian and endometrial carcinomas.
Human Epididymis Protein 4
Human epididymis protein 4 (HE4) was approved in 2009. It is recommended for monitoring patients for recurring epithelial ovarian cancer. Disease recurrence or progression can be indicated if HE4 levels are ≥150.1 pM. This marker is not specific for ovarian cancer. Therefore, it is not suitable for use in the screening or diagnosis of ovarian cancer.
Thyroglobulin
Thyroglobulin (Tg) is produced and used exclusively by the thyroid gland. A Tg assay is frequently ordered prior to thyroid surgery to determine whether the tumor is producing Tg. This assay can be performed to monitor cancer recurrence because of rising levels over time following thyroid surgery. Tg levels can be elevated not only in thyroid cancer, but in Graves’ dis ease and thyroiditis.
Prostate-Specific Antigen and Prostatic Acid Phosphatase
Prostate cancer is a leading cause of cancer death in U.S. men. Although there has been controversy in recent years about the application of prostate assays, there are two tumor markers for cancer of the prostate, prostate-specific antigen (PSA) and prostatic acid phosphatase.
Prostate-Specific Antigen. PSA screening has been controversial in recent years. Research on PSA testing offers mixed results on the benefits of PSA screening testing. However, some investigators suggest that PSA-screened men were more likely to be treated for prostate cancer at academic centers, where they got more state-of-the-art treatment.
PSA is a prostate tissue–specific marker, but not a prostate cancer–specific marker. It is a protease enzyme secreted almost exclusively by prostatic epithelial cells. Blood levels of PSA are increased when normal glandular structure is disrupted by benign or malignant tumor inflammation. The serum PSA level is directly proportional to tumor volume, with a greater increase per unit volume of cancer compared with benign hyperplasia. However, elevated PSA levels can be detected in prostate infection, irritation, benign prostatic hypertrophy, and recent ejaculation.
Free PSA assists in distinguishing cancer of the prostate from benign prostatic hypertrophy (BPH). Comparison of free PSA to PSA levels is used to assess the risk of cancer because the ratio of free PSA to PSA in prostate cancer is decreased. PSA levels appear useful for monitoring progression and response to treatment in patients with prostate cancer.
Other techniques that have been used for the detection of prostate cancer include PSA velocity (incremental increase of PSA over time), PSA density (ratio of serum PSA to prostate volume), age-adjusted PSA (PSA increases with age), biostatistically derived algorithms, free and total PSA, complexed PSA and, most recently, human kallikrein II, a molecule similar but not identical to PSA.
Other Prostate Cancer Biomarkers. Prostatic acid phosphatase is another older marker for prostate cancer. It is a serum enzyme exclusively diagnostic of prostatic carcinoma.
Isoforms of PSA represent the next generation of prostate cancer detection. An alternative marker is hK2. Its serum level is also elevated in men with prostate cancer. Another marker, early prostate cancer antigen-2 (EPCA-2), can specifically identify prostate cancer and distinguish aggressive from nonaggressive disease. These newer PSA-based screening assays will also be helpful for diagnosis and monitoring treatment.
Carcinoembryonic Antigen
The cell surface protein carcinoembryonic antigen (CEA) is found predominantly on normal fetal endocrine tissues in the second trimester of gestation. If CEA is detected in mature individuals, it is of limited diagnostic value but is helpful in differentiating between benign and malignant pleural and ascites effusions. CEA was first described in 1965 as a tumor marker specifically elevated in patients with colon cancer; it was later found to be elevated in patients with breast, lung, liver, and pancreatic cancers. Plasma levels higher than 12 ng/mL are strongly correlated with malignancy. Elevated neoplastic states frequently associated with an increased CEA level are endodermally derived gastrointestinal neoplasms and neck and breast carcinomas. Also, 20% of smokers and 7% of former smokers have elevated CEA levels.
CEA is used clinically to monitor tumor progress in patients who have diagnosed cancer with a high blood CEA level. If treatment leads to a decline to normal levels (<2.5 ng/mL), a rise in CEA level may indicate cancer recurrence to the clinician. A persistent elevation is indicative of residual disease or poor therapeutic response. In patients who have undergone colon cancer resection surgery, the rate of clearance of CEA levels usually return to normal within 1 month, but may take as long as 4 months. Blood specimens should be obtained 2 to 4 weeks apart to detect a trend.
CA 19-9
CA 19-9 is a glycolipid, Lewis blood group carbohydrate. Elevated levels have been found in patients with pancreatic, hepatobiliary, colorectal, gastric, hepatocellular, pancreatic, and breast cancers. Its main use is as a marker for colorectal and pancreatic carcinoma. This marker has greater specificity for pancreatic cancers than CEA. CA 19-9 is also known as gastrointestinal cancer–associated antigen.
CA 15-3
CA 15-3 is a biomarker used in conjunction with patient history, physical examination, and mammography during active cancer therapy to monitor metastasis. CA 15-3 is a high molecular-weight (HMW) glycoprotein coded by the MUC II gene and expressed on the ductal cell surface of most glandular epithelial cells. The main purpose of the assay is to monitor patients after mastectomy. Using a cutoff of 25 U/mL for CA 15-3, the detection rate is only 5% for stage I breast cancer.
The sensitivity is much better in higher stage disease, which makes it a good measure of tumor burden. CA 15-3 is positive in other conditions, including liver disease, some inflammatory conditions, and other carcinomas. A change in the CA 15-3 concentration is more predictive than the absolute concentration. Over time, tumor markers exhibit a steady state in the body, a balance between antigen production by the tumor and degradation and excretion. Changes in tumor burden are reflected by changes in the tumor marker concentration.
A high CA 15-3 level (>32 U/mL) usually indicates advanced breast cancer and a large tumor burden. This bio marker lacks sensitivity and specificity and is approved only for monitoring patient response to treatment and recurrence.
CA 27.29: Breast Carcinoma–Associated Antigen
Carcinoma of the breast often produces mucinous antigens that are HMW glycoproteins with O-linked oligosaccharide chains. Monoclonal antibodies (MAbs) directed against breast carcinoma–associated antigen (CA 27.29) can quantitate the levels of this antigen in serum. The antibodies recognize epitopes of a breast cancer–associated antigen encoded by the human MUC1 gene, which is also referred to as MAM6, milk mucin antigen, CA 27.29, and CA 15-3. This tumor marker may be useful in conjunction with other clinical methods for predicting early recurrence of breast cancer. It is not recommended as a breast cancer screening assay. Increased levels of CA 27.29 (>38 U/mL) may indicate recurrent dis ease in a woman with treated breast carcinoma and may indicate the need for additional testing or procedures. Some clinical investigators do not endorse the routine use of this new marker.
HER2/neu
HER2/neu is encoded by an oncogene and is over expressed in 15% to 20% of invasive breast cancers. It is associated with increased tumor aggressiveness and a reduced survival rate. This biomarker is a predictive assay to assess tumor susceptibility to therapy, such as lapatinib and trastuzumab (Herceptin, a humanized monoclonal antibody that targets HER2/neu).
Other Cancer Biomarkers
β-Human Chorionic Gonadotropin (β-Beta Subunit) β-hCG, an ectopic protein, is a sensitive tumor marker with a metabolic half-life in vivo of 16 hours. A serum level of β-hCG higher than 1 ng/mL is strongly suggestive of pregnancy or a malignant tumor such as an endodermal sinus tumor, teratocarcinoma, choriocarcinoma, molar pregnancy, testicular embryonal carcinoma, or oat cell carcinoma of the lung.
Miscellaneous Enzyme Markers
Lactic dehydrogenase (LDH) is a frequently measured enzyme of the glycolytic pathway. The level of LDH is elevated in a wide variety of malignancies and other medical dis orders. Its level has been shown to correlate to tumor mass in solid tumors so it can be used to monitor progression of these tumors.
Neuron-specific enolase is an isoenzyme specific for all tumor cells derived from the neural crest. An enzyme increase has been detected in neuroblastoma, pheochromocytoma, oat cell carcinomas, medullary thyroid and C cell parathyroid carcinomas, and other neural crest–derived cancers. Serum levels are frequently elevated in disseminated disease.
Placental alkaline phosphatase (ALP) can be detected during pregnancy. ALP is also associated with the neoplastic conditions of seminoma and ovarian cancer.
Miscellaneous Hormone Markers
Elevated or inappropriate serum levels of hormones can function as tumor markers. Adrenocorticotropic hormone (ACTH), calcitonin, and catecholamines may be secreted by differentiated tumors of endocrine organs and squamous cell lung tumors. Oat cell carcinomas may produce β-hCG, antidiuretic hormone (ADH), serotonin, calcitonin, parathyroid hormone (PTH), and ACTH. These hormones can be used to follow a patient’s response to therapy.
In addition, some breast cancers demonstrate progesterone and estradiol (estrogen) receptors, which are strongly correlated with a positive response to antihormone therapy. Patients with neuroblastoma and pheochromocytoma secrete catecholamine metabolites that can be detected in the urine. Neuroblastomas also release neuron-specific enolase and ferritin; these markers can be used for diagnosis and prognosis.
Breast, Ovarian, and Cervical Cancer Markers
For more than 15 years, circulating breast cancer antigens have been used to monitor therapy and evaluate recurrence of the cancer. Estrogen and progesterone receptors are universally accepted as prognostic markers and therapeutic choice indica tors. A relatively new approach has been the use of the oncogene HER2/neu as a prognostic indicator and a marker related to the choice of therapy. This has been particularly useful since the introduction of trastuzamab as a chemotherapeutic agent that targets the HER2/neu receptor. Breast cancer patients who express HER2/neu in their cancers have a poor prognosis with shorter disease-free and overall survival than patients who do not express HER2/neu. The evaluation of HER2/neu has two clinical functions: (1) predictive marker for response to trastuzumab therapy; and (2) prognostic marker.
A newer and more powerful predictor of the outcome of primary breast cancer in young women has been reported. Microarray analysis of a previously established 70-gene profile has demonstrated that a good prognosis gene expression signature is a strongly independent factor in predicting disease outcome.
Epidermal Growth Factor Receptor
EGFR and human epidermal growth factor receptor-2 (HER 2, HER2/neu, or c-erB-2) are both transmembrane tyrosine kinase receptors expressed on normal epithelial cells but over expressed in some cancer cells. A portion of both receptors is released from the cell surface and circulates in normal people and in abnormally high levels in cancer patients. The shed portions can be measured in serum or plasma using antibody based immunoassays. These assays allow real-time assessment of the patient’s HER2/neu or EGFR status and repeat testing for patient monitoring; they can be performed in a standardized and quantitative manner.
HER2 and EGFR have been the targets of considerable pharmaceutical activity to develop therapies that will interfere with the oncogenic potential of these growth factor receptors. These therapies include small-molecule inhibitors designed to target and block the function of HER2 protein overexpression. One drug, trastuzumab, is a humanized antibody that targets cells that overexpress the HER2/neu and has been successfully used in combination with chemotherapy to increase the efficacy of the antibody-based treatment. An anti-EGFR antibody known as IMC-225 is directed against cells that overexpress the EGFR oncoprotein.
Molecular Diagnosis of Breast Cancer
The assessment of DNA content (aneuploid, diploid) and cell cycle analysis (G0G1, S, G2, M) can be of prognostic use in certain solid tumors (e.g., breast cancer). Cell cycle analysis can be performed on fresh or frozen tissue. In breast cancer, research has indicated that low S phase and diploid DNA content are associated with a relatively good prognosis; a high S phase number of cells and aneuploid DNA content have a tendency to indicate a worse prognosis. The DNA content of a tumor is classified in order of worsening prognosis from diploid, near-diploid, tetraploid, aneuploid, hypertetraploid, and hypoploid. The ratio of tumor G0G1 DNA content to normal G0G1 DNA con tent is called the DNA index. Ploidy status and the S phase fraction should be combined with other indicators (e.g., hormone receptor status) to evaluate treatment options and prognosis.
In June 2011, the FDA approved the Inform Dual ISH, a genetic test developed by a Roche affiliate (Ventana Medical Systems, Tucson, Ariz). This test helps determine whether breast cancer patients are HER2-positive, which makes them candidates for trastuzumab therapy. The Dual ISH test was designed to detect amplification quantitatively by light micros copy of the HER2 gene using two-color chromogenic in situ hybridization (ISH) in formal-fixed, paraffin-embedded human breast and gastric cancer. An advantage of this procedure is that it is possible to view HER2 and chromosome 17 signals directly under a microscope and for a longer period.
Bladder Cancer
Bladder cancer tumor markers for the management of patients with bladder cancer have been actively investigated. Assays approved for clinical use include the following:
• Matritech nuclear matrix protein (NMP-22)
• Bard’s BTA test
Almost all human tumors contain telomerase, a growth enzyme that promotes the malignant proliferation of cancer. Normal cells usually do not have this enzyme, but telomerase renews the DNA of tumor cells and permits indefinite replication.
Telomerase was first observed in ovarian cancer cells and its presence was later established in almost all cancers. It is not clear whether other vital cells need telomerase to function. For example, telomerase inhibition could adversely affect stem cells, which help produce blood cells and lymphocytes and may need the enzyme to function. Second, telomerase inhibition has not been proved or tested physiologically in human beings. Finally, a drug based on telomerase would have to reduce the ability of the cancer to spread. Screening for telomerase inhibitors and plans for future studies to discover and develop chemicals that block the action of telomerase may suggest a design of more effective anticancer drugs.
Monocyte Chemotactic Protein
Serum levels of a newer marker, monocyte chemotactic protein-1 (MCP-1) have been found to be helpful before and after vaccination with a HER2/neu E75 peptide plus granulocyte-macrophage colony-stimulating factor vaccine. Levels of serum MCP-1 higher than 250 pg/mL have correlated with favorable prognostic variables in breast cancer.
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