Introduction

Tumor biomarkers are substances synthesized by cancer cells or other body cells in response to cancer and released into the circulation.[1] Tumor biomarkers vary widely in structure and may be simple molecules, such as catecholamines; well-characterized proteins, such as hormones, enzymes, or gene products; or more heterogeneous glycoproteins or mucins, such as carbohydrate antigen 125 (CA 125), which may be quantified by the antibodies used to measure them. Several important tumor biomarkers such as alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), and human chorionic gonadotropin (hCG) are oncofetal antigens present in the normal fetus, expressed in minute concentrations by healthy tissues, but expressed at high concentrations by some malignancies.[2]

Assays of tumor biomarkers are employed in various clinical settings and are an integral part of many cancer diagnoses and management plans. Using various techniques, these biomarkers may be assayed in selected body fluids such as blood, urine, and pleural or peritoneal effusions. Assays of tumor biomarkers may aid in the screening and early diagnosis of malignancy, guide treatment decisions, monitor treatment response, assess disease progression, or detect cancer recurrence.[3] 

However, assays of tumor biomarkers have limitations and should not be used as standalone diagnostic tools.[4] The results of tumor biomarker assays are most effective when interpreted with clinical information, imaging studies, and pathological tissue examination to ensure a comprehensive assessment and facilitate an accurate diagnosis.

The ideal tumor biomarker would be an inherently stable molecule with high specificity, sensitivity, accuracy, and reproducibility rates, offering cost-effective screening, diagnosis, and prognostic indication. However, no clinically employed tumor biomarker possesses all these characteristics. Most tumor markers have limitations in specificity, sensitivity, or clinical utility, making it essential to use them in conjunction with other diagnostic tools for comprehensive patient evaluation and management.[1][3][5] 

Etiology and Epidemiology

Cancer is not one disease but a group of diseases characterized by dysregulated cellular growth. Malignant neoplasms have the ability to infiltrate, invade, and destroy surrounding tissues.[6] Cancers are caused by genetic mutations that may be inherited or acquired through exposure to environmental carcinogens.[7] Carcinogens like tobacco smoke, asbestos, ionizing and ultraviolet radiation, and infections are associated with malignancy development.[8] 

Cancer is one of the leading causes of death worldwide, accounting for 10 million deaths yearly.[9] On average, 22.6% of women and 18.6% of men risk developing cancer before the age of 75 years.[10] In younger patients, the most commonly encountered malignancies are hematological. However, in older populations, breast, prostate, lung, and colorectal cancers are the most common; these four malignancies comprise more than half of the cancer diagnoses made worldwide. Cancer incidence is increasing globally due to an aging population, lifestyle changes, and environmental pollution.

Bence Jones protein was the first tumor biomarker described in the literature.[11] Since then, many protein- and hormone-based tumor biomarkers have been described and introduced into clinical practice. More recently, proteomics and genomics technology have permitted the assay of various genetic and molecular tumor biomarkers using microarray techniques.

Specimen Requirements and Procedure

The National Academy of Clinical Biochemistry (NCAB) has established preanalytical quality guidelines for tumor biomarkers.[12] Assays of serum should be collected in red-top containers. Other body fluids to be assayed should be collected in fluid-specific containers.[13] Chromosomal assessment of bone marrow requires 2 to 3 mL of bone marrow from the first pull of the repositioned needle during marrow extraction.[14] Whole blood is required for microarray techniques.[15] Immunohistochemistry staining requires approximately a 1-mL tissue volume; the sample should be deparaffinized and rehydrated before staining.[16]

It is preferable to assay all samples immediately. Tissue or bone marrow samples for chromosomal assessment, fluorescent in situ hybridization, or microarray should not be frozen. Salivary contamination may cause falsely increased concentrations of CEA and carbohydrate antigen 19-9.[17] Specimens can be collected at any time of the day; no diurnal variation has been documented. Specimens should always be collected before any invasive procedures since tissue trauma may cause a transient release of tumor markers into the circulation. For example, prostate-specific antigen (PSA) levels increase following urinary catheterization and prostate biopsy, and CEA levels increase after colonoscopy. Tumor biomarker assays should be ideally repeated after 2 to 3 weeks for additional evidence.[18]

The commonly measured tumor markers are generally stable. However, serum or plasma should be separated from the clot and stored at 4 °C in the short term or below -30 °C as soon as possible; relevant guidelines should be followed if available. For prolonged storage, specimens should be stored at -70 °C.[19] Heat treatments should be avoided, such as those used to deplete serum complement components or inactivate the human immunodeficiency virus. Avoidance of heat treatments is particularly true for PSA and hCG, which may dissociate into its free α- and β-subunits at increased temperatures.[20]

Diagnostic Tests

Many malignancies have one or more tumor biomarkers that are routinely measured in the course of diagnosing, managing, and monitoring for recurrence of the neoplasm. Some of these malignancies and their respective biomarkers are listed in Table 1. Malignancies and Related Tumor Biomarkers.

Table 1. Malignancies and Related Tumor Biomarkers.

Testing Procedures

Various assays can be employed when assessing tumor biomarkers. The most commonly utilized testing procedures include:

• Enzyme assays: Most enzymatic tumor biomarkers, except PSA, are quantified using an enzyme activity assay; enzymatic activity is used to determine the amount of biomarkers in the tested sample.[1] Enzyme activity assays are performed by adding excess substrate and cofactors to the prepared sample, noting their conversion rate to the product. Alternatively, kinetic enzyme assays may be used, which measure substrate at specific, predetermined intervals. 
• Immunoassays: Immunoassays are based on the principles of the antigen-antibody reaction; when measuring tumor biomarkers, the biomarker serves as the "antigen," and antigen-specific antibodies are used. Commonly employed immunoassay methods are enzyme-linked immunosorbent assay (ELISA), electrochemiluminescence immunoassay, and immunohistochemistry. Many tumor biomarkers are quantified using immunoassay techniques, including AFP, CEA, hCG, prolactin, calcitonin, and carbohydrate antigens.[21]
• High-performance liquid chromatography (HPLC): HPLC is primarily employed when identifying catecholamines and their metabolites in plasma and urine. HPLC runs analytes over a column and separates them based on their physical properties.[22] 
• Immunohistochemistry: Immunohistochemistry is a type of immunoassay employed when tumor biomarkers must be detected in solid tissue samples obtained via biopsy. A thin tissue slice is placed on a glass slide, antibodies are added against the specific antigen, and colorimetric secondary antibodies are used to detect antigen-antibody binding. Immunohistochemistry is utilized in the detection of estrogen and progesterone receptors and Her2.[23] 
• Fluorescent in situ hybridization (FISH): FISH identifies specific genetic mutations in tumor cells.[24] This technique uses fluorescent-labeled DNA probes, which hybridize to specific target sequences in cells, allowing their visualization under a fluorescence microscope. APC (adenomatous polyposis coli) and ras mutations and Her2 overexpression are identified using FISH.[25]
• Polymerase chain reaction (PCR): PCR is a technique that amplifies a specific DNA segment by cycling through denaturation, annealing of primers, and extension using a thermostable DNA polymerase. PCR is used to detect the bcr-abl1 fusion present in many forms of chronic myeloid leukemia. PCR is also used to detect microsatellite instability or mutations in genes like K-ras, N-ras, and BRAF, which have prognostic and predictive implications in colorectal cancer.[26] PCR is utilized to detect HER2 gene amplification, which helps identify patients who may benefit from HER2-targeted therapy.
• Microarrays: Microarrays permit the identification and evaluation of multiple genetic mutations or overexpression simultaneously using a solid two-dimensional support material such as a silicon chip with multiple spots containing sequences from thousands of genes. Fluorescent-labeled complementary DNA from the specimen can hybridize, and the signal is measured. Microarrays have many applications; in oncology, they are used for genetic profiling in ovarian and colorectal cancer, genetically typing leukemias, and identifying the tumor of origin in metastatic lesions.[27]

Interfering Factors

Disadvantages of Tumor Biomarkers 

Variations in sample collection, handling, storage, and assay techniques can alter the biomarker profile in a given sample. Standardization of the preanalytical and analytical variables may mitigate these variations. Very low concentrations of tumor biomarkers are frequently encountered in early-stage tumors; a highly sensitive assay is required.[28] Tumor biomarkers also have inherent drawbacks and disadvantages, including:

• Lack of specificity: Some tumor biomarkers are produced by normal and cancerous cells and are elevated in many noncancerous conditions. This lack of specificity can lead to false-positive results, potentially leading to unnecessary diagnostic procedures or treatment interventions.[2]
• Lack of sensitivity: Tumor biomarkers may not be elevated in all individuals with a specific cancer, particularly in the early stages of the disease. This lack of sensitivity can result in false-negative results, where biomarker levels are within normal ranges despite the presence of cancer. Such a scenario may delay the diagnosis and lead to missed opportunities for early intervention and treatment.[29]
• Biological variability: Individual patients can exhibit biological variability in tumor biomarker levels, making it challenging to establish universal reference ranges or cut-off values for diagnosis or monitoring.[30] Biological factors such as age, gender, genetics, and comorbidities can influence biomarker levels, leading to variations in results among individuals.
• Analytical variability: Variations in assay platforms, reagents, and laboratory techniques can contribute to analytical variability in tumor marker measurements. Inconsistent methods and lack of standardization can impact the accuracy and reliability of results, hindering data comparison across different laboratories or over time.[31]
• Limited diagnostic utility: Tumor biomarker assays are unsuitable as standalone diagnostic tools for cancer and should be used in conjunction with other diagnostic methods, such as imaging studies, biopsies, or clinical evaluations, to establish a comprehensive diagnosis.[2] Relying solely on tumor biomarker assays may lead to incomplete or inaccurate diagnostic conclusions.

Commonly Encountered Interferences

• High-dose hook effect: This effect is characterized by falsely low values at high tumor biomarker concentration and is commonly seen in patients for whom the assay is being performed for the first time.[32] The high-dose hook effect can be avoided by using solid-phase antibodies of higher binding capacity, performing the assay in two sample dilutions, and implementing proper wash steps.
• Specimen carryover: This interference is most commonly encountered when dealing with high-concentration markers in the assay.
• Interferences from heterophilic or human antimouse antibodies: Samples from patients who have undergone monoclonal antibody therapy or have circulating anti-animal antibodies may return falsely high or low values.[33] Identifying the presence of interfering antibodies requires a high degree of clinical suspicion that a tumor marker result is incorrect; this clinical suspicion may be strengthened if pertinent clinical information is available. Once suspected, potential interference can be investigated by testing the specimen at various dilutions, retesting after treatment with a commercially available blocking agent, adding additional nonimmune mouse serum to the reaction mixture and reassaying, or by reassaying the specimen using a different method provided by another manufacturer, preferably using a different methodology.[34] Caution should be applied when interpreting these results.
• Pharmaceutical interference: Anticoagulants such as ethylenediaminetetraacetic acid (EDTA) might interfere with some assays.[35]

Results, Reporting, and Critical Findings

The reported results should include reference intervals specific to the employed method and derived from an appropriate healthy population.[36] If possible, the technique used for the assay should be mentioned when reporting results. If there has been a method or technique change, the laboratory should indicate whether it will likely influence the interpretation of the trend in results. There should be a defined protocol if methods are changed, and the likely effect should be communicated to clinical users before the change.[3] Managing the change may necessitate analyzing the previous specimen by the new method or requesting another specimen to reestablish the baseline or confirm the trend in biomarker concentrations.[37]

Instead of interpreting a single value, observing the overall trend of the biomarker concentration resulting from interval testing over time is more likely to provide valuable insight into the status of the disease. Graphical reporting can offer a clear and concise way to interpret the trend in biomarker concentrations over time.[1] Recording brief clinical information alongside the laboratory data enhances result interpretation. Recommendations on the need for confirmatory specimens and the next assay interval can also be included.

Reporting critical increases in tumor biomarker concentrations and accounting for the analytical performance of the test, biological variations, and individual reference intervals contributes to an earlier diagnosis of relapse. The percentage increase or decrease that constitutes a significant change should be defined, account for analytical and biological variation, delineate the expected rate of change in benign and malignant conditions and report the time between samples.[38] For tumor biomarkers, differences in the magnitudes of their biological variation contribute significantly to these percentages.[39]

The half-life of the tumor biomarker must be considered when interpreting test results. Before surgical intervention, use the known half-life of the biomarker to estimate the time required for the level to decline to a normal or undetectable level.[40] If the quantitative decline of a tumor biomarker will be used to determine the likelihood of complete tumor resection, biomarkers should not be measured until at least 2 weeks and ideally 4 weeks postoperatively.[1] The rate of decline may be affected by underlying comorbidities such as renal or hepatic dysfunction.[41] For example, serum CEA often remains inappropriately elevated in patients with underlying hepatic dysfunction due to inefficient hepatic metabolism of the serum biomarker.[42] Persistently elevated serum β-2 microglobulin is frequently noted in patients with acute and chronic renal disease; even the small-sized β-2 microglobulin molecule has difficulty passing through the injured glomerular apparatus.[43]

If appropriate to the specific neoplasm, clinicians should consider ordering a panel of tumor biomarkers to increase the diagnostic sensitivity and specificity of the test.[1] Many malignancies have a heterogeneous cellular composition and express more than one tumor biomarker; measurement of multiple biomarkers is frequently required to achieve a >90% detection sensitivity.[13]

Clinical Significance

Tumor biomarkers each possess some degree of clinical utility and correlate to one or several specific malignancies. Many tumor biomarkers are expressed to some degree by normal, healthy cells or tissues, and levels of circulating biomarkers can be affected by benign conditions. The sensitivity and specificity of each assay must be considered within the context of the clinical condition of the patient.[30] 

There are many national and international guidelines on the selection and utilization of tumor biomarkers. The National Academy of Clinical Biochemistry, the European Group on Tumor Markers, the American Cancer Society, the National Comprehensive Cancer Network, and the National Institute for Health and Care Excellence have established recommendations regarding the use of tumor biomarkers based on the level of available evidence. 

The clinical significance of some commonly measured tumor biomarkers is described in Table 2. The Clinical Significance of Selected Tumor Biomarkers.

Table 2. The Clinical Significance of Selected Tumor Biomarkers.

Quality Control and Lab Safety

The testing laboratory is responsible for implementing stringent quality control measures to ensure the accuracy and reliability of the test. Assays should be validated before clinical use to ensure the provision of accurate and relevant reports. Recommended intra-assay and interassay variability are <5% and <10%, respectively. Some newer techniques may perform significantly better but may be less precise.[12]

Aspects of quality control, such as internal and proficiency testing (PT), should be implemented. The quality control specimen should mimic sera, and multiple levels can be used to cover the range of concentration, including the decision limits. It is important to include negative and low-positive controls.[65] The number of internal quality control samples to be run for marker assay validation depends on the frequency of testing. The samples should be checked frequently for assay interferences. During tumor marker assay, calibration and daily maintenance should be conducted before running quality control (QC) samples.[66] 

Immediate and appropriate action should be taken to avoid erroneous reporting when an assay run fails to meet objective criteria for assay acceptance. Criteria for acceptance should be predefined and based on logical criteria such as those of Westgard. The number of IQC specimens included per run should allow the identification of an unacceptable run with a given probability appropriate to the clinical application.[67] Given the long-term monitoring of cancer care, assay stability should be ensured over prolonged periods. Laboratories should have procedures and acceptance criteria for assessing lot-to-lot variation that may adversely affect clinical outcomes.[68]

Quality control (QC) material not provided by the method manufacturer is preferable; kit controls may provide an overly optimistic impression of performance as they are unlikely to be commutable with patient serum. At least one authentic serum matrix control from an independent source should be included in addition to any QC materials provided by the method manufacturer.[66]

PT specimens should be commutable with patient specimens to ensure valid between-method comparisons.[69] Concentrations should assess performance over the working range and should include an assessment of linearity on dilution, baseline security, and stability of results over time. The PT provider is responsible for ensuring that specimens are stable in transit. The target values, usually consensus means for heterogeneous analytes, should be accurate and stable, as demonstrated by assessing their accuracy, stability, and linearity on dilution.[70]

When performing tumor biomarker assays, adhere to standard laboratory safety practices, including personal protective equipment, handling and disposal of biohazardous materials properly, and maintaining a clean work environment. Follow equipment maintenance and calibration protocols, and ensure staff is trained in emergency procedures to promote a safe and efficient laboratory environment.[71]

Enhancing Healthcare Team Outcomes

Tumor biomarker assay requires a multifaceted approach. Laboratory technicians with expertise in running tumor marker assays are essential to ensure accurate testing. Lab professionals' roles include selecting appropriate assays for specific cancer types, establishing appropriate cutoff values, and determining the significance of marker trends over time. Clinicians should have a strategy toward evidence-based practices and the clinical utility of tumor marker assays.

Healthcare providers should uphold ethical principles while discussing test results, potential limitations, and implications for treatment choices. Patient safety is of paramount importance throughout the assay process. Adequate measures should be in place to prevent contamination, ensure specimen integrity, and safeguard patient information. Interprofessional communication and coordination are crucial for seamless care. Collaboration among physicians, pathologists, laboratory technicians, and other healthcare professionals ensures accurate sample collection, timely test results, and effective integration of tumor marker data into patient management plans. This collaborative effort enhances care coordination within a concise framework.