FFPE samples are widely used in research and diagnostics because they preserve tissue morphology and cellular detail over long periods. However, the formalin fixation process can cause crosslinking between proteins and nucleic acids, which may alter protein conformation and impact detection.

To accurately analyze protein expression in FFPE samples, researchers often use techniques such as:

• Immunohistochemistry (IHC) uses antibodies to detect and visualize proteins within the tissue.
• Mass spectrometry (MS) enables proteomic profiling, albeit with more preparation.
• RT-PCR and qPCR are primarily used for mRNA and offer an indirect insight into protein levels.

The selection of antibodies, antigen retrieval methods, and sample preparation protocols significantly influences the quality and reproducibility of your results.

Cancer biomarkers help detect, diagnose, and monitor cancer. Some are specific to a single cancer type, while others appear in multiple cancers. Protein-based markers remain among the most widely used in clinical settings.

3 Common Protein-Based Tumor Markers

Protein-based cancer biomarkers are substances that tumors release into the bloodstream. Some of the most prevalent protein-based markers include:

1. Alpha-Fetoprotein (AFP)

Alpha-fetoprotein is a tumor marker used to detect and monitor liver and testicular cancer. High levels of AFP in adults often indicate hepatocellular carcinoma, the most common type of liver cancer. AFP is also used to monitor treatment response and detect recurrence. However, high AFP levels do not always mean cancer is present, which directs healthcare professionals to order additional tests for confirmation.

2. CA-125

Doctors use CA-125 tumor markers to detect and monitor ovarian cancer. Many women with ovarian cancer have high CA-125 levels, but this marker is not exclusive to cancer. Other conditions, such as endometriosis and fibroids, can cause a spike in this value. Because of this, CA-125 is used along with imaging or other diagnostic tools to produce a correct diagnosis. 

3. Carcinoembryonic Antigen (CEA)

CEA helps doctors detect and monitor colon cancer, but it’s also useful for tracking pancreatic, lung, and breast cancers. Malignant tissue usually produces more CEA than normal tissue, so rising levels can signal tumor growth or treatment response. But it’s not always a clear-cut sign of cancer. Smoking, inflammation, and other non-cancerous conditions can also raise CEA levels, which is why healthcare workers consider it alongside other tests for a more accurate diagnosis.

3 Types of Tumor Markers for Specific Cancers

Some cancer biomarkers are strongly associated with specific cancer types. Here’s a comprehensive list of common cancer biomarkers:

1. Colon Cancer Tumor Marker

Colon cancer tumor markers (CEA) indicate colon cancer. Doctors use blood tests to measure carcinoembryonic antigen (CEA) levels. Higher concentrations of CEA often indicate the presence of active disease. However, because CEA is not specific to colon cancer, its accuracy is enhanced when monitored over time rather than depending on a single measurement. A steady increase in CEA levels may suggest tumor growth or recurrence, while decreasing levels after treatment typically indicate a positive response to therapy.

2. Carcinoembryonic Antigen Tumor Marker 

Carcinoembryonic antigen (CEA) is a glycoprotein found in the fetal digestive system, but its levels drop significantly after birth. In healthy adults, CEA is typically present in very low amounts. CEA, a pancreatic cancer tumor marker, is commonly used to assess pancreatic cancer early. However, some cancers (especially colorectal cancer) can cause a sharp increase in CEA levels, making it a useful tumor marker for monitoring disease progression and treatment response.

Doctors measure CEA levels through a simple blood test. While CEA is most commonly associated with colorectal cancer, it is also used to monitor other malignancies, including pancreatic, lung, and breast cancers.

3. Hepatocellular Carcinoma Tumor Marker

Hepatocellular carcinoma (HCC), the most common type of liver cancer, is often detected using tumor markers like alpha-fetoprotein. AFP is a primary tumor marker used to detect and monitor hepatocellular carcinoma, particularly in individuals with chronic liver disease.

A predictive biomarker is measured before treatment, and it’s a characteristic that identifies individuals who are more likely to benefit from or experience a certain outcome from a particular therapy. Predictive biomarkers provide extensive information on the probability of response patients will have to a specific type of therapy, whether positive or negative, and are essential for healthcare professionals and those in scientific fields. The probability of survival depends on treatment in patients who express the biomarker associated with a response to a particular therapy. In oncology and other medical fields, predictive biomarkers are used to tailor therapies for better outcomes.

To obtain high-quality nucleic acids from FFPE tissues, researchers must optimize several key steps in the extraction process. First, effective deparaffinization is crucial to remove paraffin, which can interfere with enzymatic digestion and nucleic acid purification. This is typically achieved using xylene or alternative solvents. Second, heat-induced crosslink reversal is necessary to break DNA-protein bonds caused by formalin fixation. Applying high temperatures (194℉–208℉) improves DNA integrity and accessibility. Next, DNase treatment is often used to eliminate contaminating genomic DNA when extracting RNA, ensuring the purity needed for gene expression studies. Finally, quality control measures such as spectrophotometry, fluorometric quantification, and gel electrophoresis help assess nucleic acid integrity and concentration before further analysis.

A secondary antibody is used to detect and amplify the signal of the primary antibody. This antibody is conjugated to a detectable label, such as an enzyme or fluorophore, allowing visualization of the antigen-antibody complex. To answer this question simply, a secondary antibody is an antibody designed to recognize and bind to the primary antibody rather than the antigen itself. This step enhances signal intensity and improves the accuracy of antigen detection. 

There are various types of secondary antibodies, each suited for different detection methods. The HRP secondary antibody is conjugated to horseradish peroxidase (HRP), which catalyzes a colorimetric reaction for visualization. Alternatively, the biotinylated secondary antibody is linked to biotin, which binds strongly to avidin or streptavidin for signal amplification. In fluorescence-based detection, a fluorescent secondary antibody is conjugated to fluorophores like FITC, Alexa Flu, or Cy dyes, allowing for high-resolution imaging under a fluorescence microscope.

One of the most well-established applications of dual staining in cancer screening is cervical dual stain LBP (liquid-based cytology). This test employs two biomarkers:

• P16: A tumor suppressor protein associated with high-risk HPV infections and precancerous cellular changes. 

• Ki-67: A proliferation marker indicating active cell divisions, often found in dysplastic or cancerous cells.

When combined, these markers improved the detection of high-grade cervical intraepithelial neoplasia (CIN2+), which can progress to cervical cancer if left untreated. Unlike traditional Pap tests, which often require follow-up testing, dual stain-positive results provide immediate, actionable insights into a patient’s risk level. 

The immunostaining process begins with a careful preparation of FFPE tissue, which is sectioned into thin slices and mounted on glass slides. Before staining can occur, these sections are deparaffinized to remove the paraffin wax and rehydrated to prepare the tissue for aqueous solutions. One critical step in the process is antigen retrieval, which involves exposing the tissue to heat and a retrieval buffer to unmask antigens that may have been masked during formalin fixation. This ensures antibodies can bind effectively to their target antigens. 

A blocking agent is applied to minimize background noise, which helps reduce the non-specific binding of the antibodies after immunostaining. This is followed by the application of a primary antibody, which is selected based on the antigen of interest. For instance, researchers investigating HPV-related cancers often use a p16 immunostain, while those studying gastric conditions might choose an immunostain for Helicobacter pylori. Once the primary antibody binds to its target, a secondary antibody conjugated with a detection marker is introduced to amplify the signal. 

Detection methods vary depending on the study’s needs. Some laboratories use colorimetric systems, where a visible dye indicates the presence of the antigen, while others use fluorescence for more detailed imaging. Finally, counterstaining highlights the overall tissue morphology, making it easier to interpret results in the context of the tissue structure. 

One additional consideration is the temperature and duration of antigen retrieval. Over-retrieval can cause tissue damage or loss of antigenicity, while under-retrieval may result in weak or absent staining. Adjusting retrieval conditions for specific antibodies can significantly enhance outcomes. Similarly, using high-quality mounting mediums and coverslips ensures that the final slide is preserved for analysis without compromising the immunostaining results.

Quantitative PCR, or qPCR, is also known as real-time PCR. It’s a technique that uses fluorescent reporter molecules to measure the amount of RNA or DNA in a tissue sample. qPCR gives an additional quantitative analysis of the DNA or RNA in RT-qPCR, which we’ll explore further below.

Researchers use real-time PCR to measure gene expression, viral load, and SNP detection. It’s also a vital tool for genetic testing, pathogen detection, and disease research. In qPCR or real-time PCR, the amplification of DNA is monitored in real-time using fluorescence-based detection.

There are two primary methods for detecting PCR products in real time: 

1. Dye-Based Detection (SYBR Green): Uses SYBR Green, a fluorescent dye that binds specifically to double-stranded DNA. As DNA amplifies, the fluorescence signal increases proportionally to the DNA concentration. Dye-based detection is a simple and cost-effective method, but it may detect non-specific products such as primer dimers.
2. Probe-Based Detection (TaqMan, Molecular Beacons, Scorpion Probes): Uses fluorescently labeled probes that bind specifically to the target DNA sequence. It’s more specific than dye-based detection as it only detects the target sequence, reducing the background signal. There are 3 types of probe-based methods:
   • TaqMan Probes: This probe has a fluorophore and quencher. Fluorescence is released when DNA polymerase cleaves the probe during amplification.
   • Molecular Beacons: A hairpin-shaped probe that fluoresces when it binds to the target DNA.
   • Scorpion Probes: Similar to molecular beacons, they are integrated into the primer for faster and more efficient detection.

Both methods allow DNA quantification in real-time, but probe-based detection offers greater specificity and accuracy, making it preferable for applications like gene expression analysis and pathogen detection.

Properly procuring FFPE human tissue samples requires adherence to rigorous protocols. Below, we’ll outline the key practices necessary to ensure the highest quality and integrity of these essential materials.

1. Ethical Compliance and Donor Consent

The foundation of any tissue procurement program is ethical compliance. Ensure that all samples are collected with the informed consent of donors, following guidelines set by institutional review boards (IRBs) and regulatory bodies. Transparency in communicating the purpose of tissue collection and the intended use of samples builds trust and ensures ethical integrity. 

2. Standardized Collection Protocols

Consistency is key to obtaining reliable FFPE samples. Implement standardized collection protocols that define the following: 

• Tissue Collection: Use sterile instruments and follow aseptic techniques to prevent contamination.
• Fixation Process: Promptly immerse the tissue in neutral-buffered formalin to preserve cellular integrity. Ensure the tissue thickness is appropriate to facilitate adequate penetration of the fixative.
• Paraffin Embedding: Dehydrate the tissue through a graded series of alcohol solutions before embedding it in paraffin wax. This step protects the sample from degrading during storage.

3. Quality Control Measures

Effective tissue procurement involves rigorous quality control to ensure the samples meet research and diagnostic standards. This includes: 

• Histological Assessment: Evaluate tissue sections for proper fixation and absence of artifacts.
• Molecular Testing: Confirm that DNA, RNA, and protein integrity are preserved.
• Documentation: Maintain detailed records of sample origin, collection date, and preservation conditions.

4. Storage and Inventory Management

Proper storage conditions are crucial for preserving FFPE samples over extended periods. Store paraffin blocks at room temperature in a dry, dust-free environment. Use digital inventory management systems to track sample location and history, ensuring efficient use and minimizing the risk of sample loss.

5. Adapting to Research Needs

FFPE human tissue procurement should be tailored to meet specific research requirements. Collaborate with researchers to understand their needs, whether it involves obtaining tissue from a particular organ, ensuring compatibility with molecular techniques, or providing annotated clinical data.

Cancer biomarker testing has revolutionized the way doctors treat cancer. By identifying specific biomarkers, clinicians can tailor treatment to an individual’s unique genetic makeup, a practice known as precision medicine. This approach ensures that patients receive the most effective therapies while minimizing side effects.

For example:

• Targeted Therapies: Biomarkers like EGFR and ALK guide the use of targeted therapies for lung cancer, offering treatments designed to attack specific genetic mutations.
• Immunotherapy: Biomarkers such as PD-L1 help predict a patient’s response to immune checkpoint inhibitors, a type of immunotherapy.
• Chemotherapy Optimization: Testing biomarkers can help avoid ineffective chemotherapy regimens by identifying whether a tumor is likely to respond.