Immunohistochemistry monitoring in cancer Flashcards
What is immunohistochemistry and when is it used?
- Ab-based technique that detects antigens of interest in fixed tissue sections, often visualized by light microscopy
- IHC is used in clinical practice to help diagnose diseases or to stratify for certain treatment approaches
- Widely used in research to study antigens of interest in healthy and diseased cells
What are the steps used to do immunohistochemistry?
- Collect biopsy specimens
- Tissue preparation
a. Most common processing: prepare formalin-fixed paraffin-embedded (FFPE) tissue blocks
i. Formalin induces chemical crosslinks and basically freezes the protein in its current state and cellular composition
b. FFPE tissue blocks are sectioned into thin slices (usually 4-10 microm) using a microtome
c. Sections are then transferred to glass slides - Antigen retrieval: need to revert some of the chemical crosslinking and “retrieve” the epitopes before proceeding to IHC (by heat or enzymatic)
- Antibody binding: primary antibody selection
- Detection:
a. Secondary antibodies targeted specifically to antibody molecules from animal species (e.g. X-a-Ra-IgG)
b. Enzymatic reaction: horseradish peroxidase (HRP) converts 3,3’ Diaminobenzidine (DAB) into brown precipitate - Counterstain: enhances the contrast and facilitates observation of histological features. Hematoxylin is most often used
Why would you use immunohistochemistry?
- IHC stands out among many other laboratory tests because it is performed without destruction of histologic architecture
- Assessment of an expression pattern of an antigen of interest is possible in the context of the microenvironment
- The procedure is short, simple and cost-effective. It can thus be performed in most laboratories
- Pathologists will use a “panel” of multiple antigens to help fully classify a particular tumour
- The amount of staining, the staining pattern and the location of staining (cell, cytoplasm, nucleus or membrane) all provide information for the diagnosing pathologist
- IHC is also frequently used in basic and clinical research for exploration of biomarkers
What is a biomarker?
A biomarker is a molecule that is objectively measured and evaluated as an indicator of normal biological process, pathogenic process, or pharmacological responses to therapeutic intervention. It should be reproducible and standards have to be developed
Why do a striking number of efforts to develop a biomarker fail to make it into the clinic?
There are a lot of technical challenges when using immunohistochemistry as a biomarker
What are the challenges when using immunohistochemistry as a biomarker?
•Time from when a tissue or organ is cut off from oxygen in surgery to fixation of specimen
•Factors influencing fixation: temperature, time, penetration rate, specimen dimension, volume ratio, pH of the buffer and osmolality
•Loss of antigenicity due to longer storage
•Heat or enzyme: dependent on both the antibody and the target protein and needs to be optimized for every antibody
•Use of reliable primary Abs: high specificity, little “off-target” binding.
o Monoclonal only binds to one epitope, has little batch to batch variability and little cross-reactivity. Less sensitive, but more specific
o Polyclonal binds to multiple epitopes, has more batch to batch variability and more cross-reactivity. They are more sensitive because they target more epitopes, but less specific
• Direct detection: Directly labeled Ab
• Indirect detection: Unlabeled primary Ab and labeled secundary/tertiary Abs often more sensitive, ability to enhance your signal
• Labile nature of phosphorylated proteins: tissues have to be fixed within 60 minutes post-surgery to maintain epitopes
• Many critical steps in the manual IHC method are operator-dependent and essential to the quality of the final IHC result and its reproducibility
How has the invention of automated immunohistochemistry platforms influenced the use of immunohistochemistry?
It was a major milestone: there was no operator-dependancy anymore and it was more reproducable. o Automated IHC are often “closed” systems, so you can’t introduce variables.
Choice of method is heavily influenced by purpose of laboratory.
Manual assessment of IHC staining remains the traditional method for most diagnostic and predictive decisions in pathology. However, the process is time sensitive, laborious and subjective
What is digital pathology?
Digital pathology is a high-troughput image analysis method that has the potential to outperform normal pathology in reproducibility and precision. It is still mostly a semi-automated approach. It still requires input and training by a pathologist and thus can’t replace expert pathologists
What are the advantages of brightfield microscopy?
Chromogenic DAB staining is routine practice. The chromogens are stable, the cost is low and it requires minimal laboratory space.
What are the advantages of using immunofluorescence?
It gives a high resolution and there is a broader dynamic range to be able to perform multiplex immunohistochemistry
What is possible with chromogenic multiplex immunohistochemistry?
With chromogenic multiplex immunohistochemistry you can use multiple Abs with different colours (still HRP linked) and look at multiple antigens of interest
You are not really able to automate process –> co-expression still to be assessed by pathologist and difficult if they overlap
You can only use up to 4 colours.
The staining is often quite weak
What is the difference in method between multiplex immunohistochemistry and normal immunohistochemistry?
Instead of adding DAB, you add a TSA reagent (different fluorophores)
How does multiplex immunohistochemistry work?
- Incubate with primary antibody
- Introduce HRP-linked secondary antibody
- Incubate with TSA reagent (3-10 minutes). HRP catalyzes formation of TSA free radicals.
- TSA free radicals form covalent bonds with tyrosine residues proximal to HRP. Unbound TSA radicals form dimers that are washed away. The TSA reagent attaches all the fluorophores to the antibody-complex. This really enhances the signal.
- Microwave the antibody strip. This removes the primary-secondary antibody complex, while the fluorophores remain.
You can repeat this cycle with up to 8 targets. Scanning per color can separate co-expressed markers.
What’s the use of multiplex immunochemistry for immune “monitoring” in cancer?
It is not really used for monitoring, as monitoring means that it is done over a period of time and taking a biopsy is an invasive procedure that does not allow serial sampling. Immunohistochemistry is mostly used for investigation of pre-treatment biomarkers. There is limited information on immune status during and after treatment.
What is the cancer immunoscore?
A frequently used biomarker that looks at the tme, because the tme is very important. It is based on quantification of lymphocytes, specifically CD3 and CD8 at tumour centre (CT) and invasive margin (IM). Higher amount of T-cells gives higher scores. A higher immunoscore correlates with better survival. The technique is mostly automated. The cancer immunoscore was discovered in colon cancer.
