Multiplexing IHC/IF with Digital Imaging

By: Jen Freeland, MS, HTL(ASCP)

The ability to multiplex diagnostic assays is a distinct technological trend that was highlighted during the 45th Annual Symposium of the National Society of Histotechnology (NSH). Multiplex immunofluorescence (mIF) or multiplex immunohistochemistry (mIHC) combined with digital imaging has been explored by pharmaceutical researchers for years as they study and develop targeted therapies in oncology. Now it appears mIF/mIHC assays are moving from conventional research into clinical testing. The potential clinical applications were highlighted in several different workshops at NSH this year.

Multiplexing with immunohistochemistry or immunofluorescence is achieved when multiple target antigens are marked in the same tissue specimen on the same slide. Although the development of mIF/mIHC assays can be challenging, when coupled with digital pathology, interpretation of the data can be applied to solve tough clinical problems. Pathologists can review multiple biomarkers in the same slide all at once or can apply filters to limit the view to one biomarker; this allows for a significant amount of data to be generated on a small amount of tissue (4). In cases where late-stage cancer is being evaluated by needle core biopsy or fine needle aspiration, the ability to garner essential clinical data from sparse starting material becomes important for maintaining tissue stewardship. These systems also can be used to quantify cellular subtypes and to study spatial relationships between a tumor and its surrounding environment (4). This information may ultimately become useful in determining which patients are candidates for different targeted immunotherapies (4). Rather than a one-test-one-drug model, multiplexing enables more of a one-test-panel-drug approach to treatment selection.

For example, in one multiplex assay (Opal® by Perkin Elmer) five biomarkers (Cytokeratin, CD4, CD68, PD-1, and PD-L1) are tagged and stained with tyramide-fluorophore chemistry in which each antibody fluoresces a different color against a DAPI nuclear counterstain (4). Multiplexed immunofluorescence delivers a lot of information on one slide, and the use of digital imaging is often utilized by pathologists to aid in the interpretation of that information (4). In the case of the above-mentioned panel, each of the antibodies can be viewed independently using different channels on the digital system. The images can also be united into one image to review proximity and co-localization of all the biomarkers (4).

Immunotherapy is another application that has utilized mIF/mIHC. Monoclonal antibodies were developed for treating cancers with certain characteristics in their immune checkpoint molecules (CTLA-4, PD-1, PD-L1), and have shown success in treating patients (3,5). But there remains a percent of the patient population in which an enduring response to these therapies is lower than desired (3). Researchers and clinicians are discovering that not only does the expression of the specific target biomarker (e.g. PD-L1 or CTLA-4) dictate whether a patient is a candidate for an immunotherapy, but the biomarker character of the tumor’s surrounding microenvironment also matters (3,10). Immune cell characteristics and their proximity to the tumor can play a role in the effectiveness of targeted immune therapies (4). Therefore, it has become increasingly important to identify a variety of immune-cell biomarkers as well as the target biomarker (such as PD-L1) and study relative quantities, types, and co-localizations (4). The most effective way to achieve this today is through mIF / mIHC.

Specifically, with immunotherapy, the presence of tumor infiltrating lymphocytes (TILs) tends to be connected to improved clinical outcomes (8). Being able to characterize TILs through mIHC and/or mIF in comparison to tumor proximity lends additional information to the pathologist than just PD-1 or PD-L1 alone (6,7). For example, the presence of B-cells (CD20+) within a tumor region is linked to improved overall survival (8). With the success of mIHC and mIF elucidating information about a tumor’s microenvironment, the technique is being expanded and applied to a wide variety of cancers.

In pharmaceutical research laboratories, mIF and mIHC are utilized in selection of candidates for clinical studies (7). At GlaxoSmithKline two mIF panels are being studied and applied to different clinical tests (7). One panel labels tissue with CD45RO, PD1, CD3, pan-cytokeratin and SOX10, and another panel labels tissues with CD8, CD68, PDL1, pan-cytokeratin and SOX10 (7). Bristol-Myers Squibb researchers have validated an mIF panel to study many cancer types, including non-small cell lung carcinoma, Merkel cell carcinoma, head & neck squamous cell carcinoma, and melanoma (1). AstraZeneca researchers recently built and validated multiplex panels for research and clinical testing (11). The advantages of multiplexed IF include the ability to identify phenotypes using multiple markers, the capacity to provide spatial information about a tumor, the illustration of morphology at both the tissue and the cellular level, and the enablement of immune profiling of solid tumors (11).

Some assays have already transitioned from clinical research and are being utilized in clinical trials. MD Anderson Cancer Center is currently enrolling candidates into a clinical trial (Study #PA19-0460) in which multiplex IHC assays will play a central role for the analysis and treatment of melanoma (12). The results of Study 1616P have been published in the Annals of Oncology, in which multiplexed IF was utilized to study gastrointestinal stromal tumors (GIST), their microenvironment, and the influence of treatment with tyrosine kinase inhibitors (13). The mIHC panel in the GIST study included biomarkers for CD3, CD8, FoxP3, PD-L1, PD-1 and DOG1 (13). Leidos Biomedical Research, a research organization affiliated with the National Institutes for Health (NIH) and the National Cancer Institute (NCI), has developed a quantitative multiplex IF assay to detect and measure activated cytotoxic T-cells (2,9). This assay was developed in collaboration with Ultivue and is designed as a companion test to inform about the effectiveness of immunotherapy, as detected by PD1 and / or PDL1 (2,9). Researchers at Leidos presented some of the assay’s results through a poster at the 2019 American Society of Clinical Pathology symposium (2,9). The assay, which identifies b-catenin, CD8, Zap70-pY493 and CD3-pY142 is being now used in multiple clinical trials (2,9).

The studies highlighted above and presented at the NSH symposium workshop illustrate the potential clinical utility of multiplexed immunohistochemistry and multiplexed immunofluorescence coupled with digital imaging to provide critical information to solve difficult clinical problems. We are fortunate that NSH encourages early collaboration and discussion through these educational sessions. Although the assays presented are research techniques today, as they continue to be utilized in clinical trials, we may see a quick transition into clinical diagnostics and into our clinical laboratories.


1. Baradet T. (2019). Validation and analysis of highly multiplexed IHC by digital analysis. 45th National Society of Histotechnology Annual Symposium. New Orleans, LA.

2. Fino K and Downing S. (2019). Investigating targeted CD3 activation in cytotoxic T cells via immune checkpoint inhibition with an advanced multiplex immunofluorescent assay. LabTools Webinar Series. November 1, 2019. https://globalmeet.webcasts.com/viewer/event.jsp?ei=1263102&tp_key=0062bdd52a

3. Gorris MAJ, et al. (2017). Eight color multiplex immunohistochemistry for simultaneous detection of multiple immune checkpoint molecules within the tumor microenvironment. The Journal of Immunology. Doi: 10.4049/jimmunol.1701262

4. Hofman P, et al. (2019). Multiplexed immunohistochemistry for molecular and immune profiling in lung cancer – just about ready for prime-time? Cancers. 11(283). https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6468415/

5. Hou Y, et al. (2018). Evaluation of immune reaction and PD-L1 expression using multiplex immunohistochemistry in Her2-positive breast cancer: the association with response to ant-Her2 neoadjuvant therapy. Clinical Breast Cancer. 18(2): e237-244. Doi: 10.1016/j.clbc.2017.11.001

6. Krull D and Downing S. (2019). Multiplex IF using DNA barcoded primary antibodies and complementary fluorophore-labeled probes: alignment of PD-L1 and PD-1 biomarker panels with true reference H&E. 45th National Society of Histotechnology Annual Symposium. New Orleans, LA.

7. Lu S, et al. (2019). Comparison of biomarker modalities for predicting response to PD-1/PD-L1 checkpoint blockade: A systemic review and meta-analysis. JAMA Oncology.5(8): 1195-1204. Doi:10.1001/jamaoncol.2019.1549

8. Monette A, et al. (2019). Immune enrichment of non-small cell lung cancer baseline biopsies for multiplex profiling define prognostic immune checkpoint combinations for patient stratification. Journal of ImmunoTherapy of Cancer. 7(86). Doi:10.1186/s40425-019-0544-x

9. Navas T, Fino K, et al. (2019). A multiplex immunofluorescent assay to assess immune checkpoint-targeted CD8 activation and tumor co-localization in FFPE tissues. Leidos Biomedical Research, Frederick National Laboratory for Cancer Research. Poster Sessions. American Society of Clinical Pathology, 2019. https://cache.webcasts.com/content/pgic001/1263102/content/0062bdd52a3db4e28ad0f09e7c865a28eec0cc6e/ASCO2019-Leidos-A%20multiplex%20immunofluorescence%20assay%20to%20assess%20immune%20checkpoint%20inhibitor-targeted%20CD8%20activation%20and%20tumor%20co-localization%20in%20FFPE%20tissues.pdf

10. Parra ER, et al. (2017). Validation of multiplex immunofluorescent panels using multi spectral microscopy for immune-profiling of formalin-fixed and paraffin-embedded human tumor tissues. Scientific Reports. 7(13380) doi:10/1038/s41598-017-13942-8

11. Surace M and Smith A (2019). Validation and analysis of highly multiplexed IHC by digital pathology. 45th National Society of Histotechnology Annual Symposium. New Orleans, LA.

12. Validation of the multiplex IF/IHC assay on surgical pathology specimens from patients with advanced melanoma and other cancer types/ Study #PA19-0460, MD Anderson Cancer Center. Currently enrolling. ClinicalTrials.gov Identifier: NCT04150562. https://clinicaltrials.gov/ct2/show/NCT04150562?rank=1

13. Yoo C, et al. Quantitative multiplexed immune profiling of advanced gastrointestinal stromal tumors (GISTs): Impact of tyrosine kinase inhibitors on microenvironment. Annals of Oncology. Volume 29, Supplement 8. Study 1616P. October 2018.

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