What is it about?

There is a plethora of information embedded in a tissue section that the conventional IHC understands only partially. Predictive biomarkers for precision immuno-oncology heavily dependent on the spatial arrangement of cells and the co-expression patterns in the tissue sections. Here we have explored the versatility of indirect multiplex immunofluorescence (mIF) and indirect multiplex immunohistochemistry (mIHC) for the labeling of breast cancer prognostic markers in routinely processed, formalin-fixed paraffin-embedded (FFPE) tissues at high resolution. The multiplex immunohistochemistry protocol utilized sequential staining for the chromogenic immunolabelling of Estrogen Receptor α (ERα) or Progesterone Receptor (PR), Human Epidermal Growth Factor Receptor 2 (HER2), and Nucleoside diphosphate kinase 1 (NM23) by multicolor chromogens in different combinations. A feasible workflow for multiplex immunofluorescence was also effectively standardized for ERα, PR, and HER2 using combinations of commercially available Alexa Fluor and Quantum dots semiconductor nanocrystal conjugated secondary antibodies. Multiplex chromogenic immunolabeling revealed differential expression of the markers on the same slide. Kappa statistics revealed perfect agreement with uniplex immunohistochemistry. For multiplex fluorescence approach, surface receptor detection using Quantum dots and Alexa fluor dyes for cytoplasmic or nuclear markers performed well for profiling multiple co-localized biomarkers on a single paraffin tissue section. The technique developed reveals additional information such as coexpression, spatial relationships, and tumor heterogeneity, providing a deeper insight into developing combinatorial therapeutic strategies in clinical care. This high throughput workflow complements the outcomes of traditional IHC while saving tissue, time, labour, and reagents.

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Why is it important?

A multiplex immunohistochemistry technique has been developed

Perspectives

The multiplex imaging offers co-expression of multiple biomarkers in tumor cells and intra-tumor heterogeneity in tumour tissues and microenvironment, which may provide prognostic significance, saving tissue samples and turnaround time. The technique described in the study can very well be followed in routine pathology practice.

Dr.K. Sujathan

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This page is a summary of: A multiplex immunoprofiling approach for detecting the co-localization of breast cancer biomarkers using a combination of Alexafluor - Quantum dot conjugates and a panel of chromogenic dyes, Pathology - Research and Practice, January 2024, Elsevier,
DOI: 10.1016/j.prp.2023.155033.
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