Can Immunohistochemistry Assays Be Standardized for Cancer Detection?

New synthetic immunohistochemistry reference standards could enable semiquantitative tumor analysis for emerging immunotherapies

Photo portrait of Zahraa Chorghay
Zahraa Chorghay, PhD
Photo portrait of Zahraa Chorghay

Zahraa Chorghay, PhD, specialized in neuroscience during her undergraduate (University of Toronto) and doctoral studies (McGill University). She continues to explore her passion for neuroscience and for making science accessible and inclusive.

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Published:Feb 03, 2023
|5 min read
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When it comes to cancer diagnostics and decision-making, clinical labs have several approaches available to them, from traditional cell culture and immunohistochemistry (IHC) to emerging molecular diagnostics, including PCR- and sequencing-based methods. Pathologists continue to rely on IHC as the gold standard for cancer detection, despite challenges regarding its standardization. However, new synthetic reference standards could enable IHC assays to provide increasingly more reliable and quantitative readouts to better inform clinicians and researchers about the presence of cancerous cells and even predict treatment response.

How does immunohistochemistry work? How is it useful in detecting cancer tissues?

IHC is a widely used technique for detecting proteins expressed by a given cell, whereby you expose the tissue specimen to antibodies that bind to specific target epitopes (or proteins). Since cancer cells mimic cells in the body, typically expressing similar epitopes as the healthy cells from which the cancer originated, researchers can use IHC to identify proteins unique to cancerous cells, thus helping pathologists classify a cancer.

IHC can provide clinically meaningful results quickly and cost-efficiently, especially with the broad adoption of automated platforms. With IHC, and relatedly, in situ hybridization, you can localize proteins to specific cells, so you can histologically identify cancer cells, allowing you to work with relatively small samples. In contrast, most molecular assays cannot pinpoint whether the detected changes were from normal or cancer cells. Moreover, unlike molecular assays, IHC can be performed on various specimen types, regardless of the preanalytical treatment (fixative). These advantages equip clinical labs to classify tumors and assess biomarkers relevant to molecular-targeted therapies with IHC.

Why is standardizing immunohistochemistry difficult?

In IHC, it is difficult to ensure consistent immunostaining for epitope detection. Different labs often report different results even from the same sample. For instance, interlaboratory error rates for the detection of HER, ER, and PER in the same paraffin block tested at two labs varied from 1.6 percent to as high as 75 percent. 

Breast cancers are tested for the presence of human epidermal growth factor receptor (HER), estrogen receptor (ER), and progesterone receptor (PER). Cancers that are positive for these markers can be targeted with inhibitory therapy.

What makes a good control in an immunohistochemistry assay?

While automation has improved the reliability of IHC, there are no reference standards, so each clinical lab chooses its own controls. According to Matthias Szabolcs, MD, director of the IHC laboratory at the Presbyterian Hospital and Columbia University Irving Medical Centre in New York, “Instead of selecting very high positive controls that will work with most protocols, labs should choose low positive controls. This allows you to correlate the signal intensity of your reaction product with the amount of target molecule in the sample, within a linear range of detection.”

He provides the example of folate receptor alpha (FOLR1) as an ideal control. Normally, FOLR1 is highly expressed in the epithelial cells along the brush border of the fallopian tube but is barely detectable in ovarian epithelial cells. This gradient of FOLR1 expression within the same tissue makes it easy to spot FOLR1 overexpression. Individuals that have epithelial ovarian cancer with high FOLR1 expression can be treated with a FOLR1-targeted treatment; the FDA recently approved both the treatment and accompanying FOLR1 diagnostic test from Roche. While FOLR1 immunostaining assays can help standardize IHC-based detection in ovarian cancer, this type of graded endogenous expression rarely applies to other cancer markers.  

Table comparing tissue, cell, and synthetic controls across 4 criteria. The first row shows images of representative controls. In the first column: a tissue with clusters of different cell types and immunostained in magenta. Second column: cell culture showing different cells immunostained in red. Third column: synthetic controls are beads. Remaining rows are comparing the three controls on the following criteria: availability of positive and negative controls; can be purchased; affordable; reliable. Tissue controls meet first 3 criteria but not lost. Cell line controls meet first 2 criteria only. Synthetic controls meet all 4 criteria.
Comparison of three types of immunohistochemistry controls.
Today's Clinical Lab

What is needed to standardize immunohistochemistry?

In a nutshell, a huge barrier to standardizing IHC has been the unavailability of reference standards. “It would be great progress in immunohistochemistry if one had these controls standardized, and if one could rely on these controls to be distributed in large quantities and with consistent immune reactivity,” says Szabolcs. 

But this is difficult to do with tissue controls, which have high variability. Alternatively, labs can use cultured cells that are engineered to express different levels of a target molecule as controls. Szabolcs explains that while there are some commercial companies that already do this, e.g., the human papilloma virus (HPV) analyte control kit from Sigma-Aldrich, using cultured cells as controls can be laborious, difficult to reproduce, and expensive.

New synthetic reference standards could enable IHC assays to provide increasingly more reliable and quantitative readouts to better inform clinicians and researchers about the presence of cancerous cells.

Hand holding a glass slide with synthetic beads labelled 1 to 10, in order of increasing concentration. Slide label reads "ER SP1 IHCalibrators."
New, synthetic reference standard involves a glass slide housing ten beads at increasing epitope concentration.

What is the new approach to immunohistochemistry reference standards?

To circumvent the issues with tissue and cell controls, Boston Cell Standards has pioneered a different approach to reference standards, which recently received FDA 510(k) clearance for evaluating breast cancers. In this approach, the target epitope is produced and loaded onto beads at specified concentrations, providing a consistent control with which to correlate the immunostaining signal intensity. When Szabolcs and his team tested these controls, beginning with ER and PER in breast cancer, they found that the slides reliably allowed the signal intensity of the samples to be compared to the linear range of target epitope expression.

How will standardizing immunohistochemistry impact cancer detection?

IHC assays, along with other assays, can be used to determine whether the patient is a good candidate for a specific therapy; this is called complementary testing. For example, programmed death-ligand 1 (PD-L1) is a predictive biomarker for patients who will benefit from PD-L1–directed immunotherapy in several malignancies, such as melanoma, and lung, kidney, and bladder cancers, and more. However, there is a lack of agreement across studies regarding the appropriate threshold of target expression for a given therapy, such as in the case of PD-L1, which is further complicated by varied expression across tumor types. 

A huge barrier to standardizing IHC has been the lack of reference standards.

According to Szabolcs, using reference standards would lead to consensus across studies on reliable thresholds for identifying patients that could benefit from immune checkpoint blockade (ICB) therapy. With improved standardization, “IHC assays would not only provide qualitative assessment about the presence or absence of an epitope, but also at least a semiquantitative assessment of the signal intensity that can be used for complementary testing of new therapies,” he says. 

Standardizing immunohistochemistry can empower labs

Establishing controls and reference curves by adopting synthetic anatomic pathology controls like those from Boston Cell Standards, alongside advances in artificial intelligence and the emergence of fully quantitative assessment through photometric analysis, would enable more accurate IHC assays. Moving forward, this will empower IHC labs to provide powerful insights for cancer diagnosis, prognosis, and therapeutic decisions.

Top Image:
Pathologists continue to rely on IHC as the gold standard for cancer detection, despite challenges regarding its standardization.
iStock, sanjeri