Validation of an Accurate Automated Multiplex Immunofluorescence Method for Immuno-Profiling Melanoma

Yaseen, Zarwa; Gide, Tuba N.; Conway, Jordan W.; Potter, Alison; Quek, Camelia; Hong, Angela; Long, Georgina V.; Scolyer, Richard A.; Wilmott, James S.

doi:10.3389/fmolb.2022.810858

Cited by 15 publications

(12 citation statements)

References 33 publications

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“…The results showed that mIF accurately detected the expression of immune cell markers (CD8, CD68, and CD16), immune checkpoint PD-L1, and melanoma marker SOX10, with better accuracy and reproducibility than IHC and single-plex IF. This study demonstrates the potential for the use of mIF in clinical practice 88 .…”

Section: Detection Methodsmentioning

confidence: 70%

Recent Advancement of PD-L1 Detection Technologies and Clinical Applications in the Era of Precision Cancer Therapy

Zhang¹,

Wu²,

Zhao³

et al. 2023

J. Cancer

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Programmed death-1 is a protein found on the surface of immune cells that can interact with its ligand, programmed death-ligand 1 (PD-L1), which is expressed on the plasma membrane, the surface of secreted cellular exosomes, in cell nuclei, or as a circulating soluble protein. This interaction can lead to immune escape in cancer patients. In clinical settings, PD-L1 plays an important role in tumor disease diagnosis, determining therapeutic effectiveness, and predicting patient prognosis. PD-L1 inhibitors are also essential components of tumor immunotherapy. Thus, the detection of PD-L1 levels is crucial, especially in the era of precision cancer therapy. In recent years, innovations have been made in traditional immunoassay methods and the development of new immunoassays for PD-L1 detection. This review aims to summarize recent research progress in tumor PD-L1 detection technology and highlight the clinical applications of PD-L1.

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Section: Detection Methodsmentioning

confidence: 70%

Recent Advancement of PD-L1 Detection Technologies and Clinical Applications in the Era of Precision Cancer Therapy

Zhang¹,

Wu²,

Zhao³

et al. 2023

J. Cancer

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“…Furthermore, in contrast to IF multiplex IHC, the development of assays involving BF multiple IHC chromogenic substrates presents some critical issues such as the visual contrast compatibility of chromogens and cellular localization of combined markers. IF multiplex IHC is undoubtedly a very powerful tool that allows a more complete and more in-depth characterization of the TME than the BF multiplex IHC [ 19 , 21 , 29 ]. IF multiplex IHC permits colocalizing more markers on the same cells than is technically impossible with BF multiplex IHC.…”

Section: Discussionmentioning

confidence: 99%

“…IF multiplex IHC permits colocalizing more markers on the same cells than is technically impossible with BF multiplex IHC. In addition, the large number of dyes available and the possibility of “turning them on” and “turning them off” coupled with automated rapid imaging system LED technologies [ 23 ] make the IF multiplex IHC the leading staining technique in the research field [ 25 , 29 ]. For all these reasons, BF multiplex IHC has remained less widespread than IF.…”

Section: Discussionmentioning

confidence: 99%

“…On the other hand, BF IHC remains the gold standard for histological evaluations [ 29 ], and the development of BF multiplex IHC could be very useful for challenging specimens such as pigmented melanoma samples. The development of standardized BF multiplex IHC protocols could be more easily implemented in the diagnostic routine workflow since pathologists are more familiar with the evaluation of stained sections in BF than IF [ 30 ].…”

Section: Discussionmentioning

confidence: 99%

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Bright-Field Multiplex Immunohistochemistry Assay for Tumor Microenvironment Evaluation in Melanoma Tissues

Ugolini

Pasqualini

Simi

et al. 2022

Cancers

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The tumor microenvironment (TME) plays a crucial role in melanoma development, progression and response to treatment. As many of the most relevant TME cell phenotypes are defined by the simultaneous detection of more than two markers, the bright-field (BF) multiplex immunohistochemistry (IHC) technique has been introduced for the quantitative assessment and evaluation of the relative spatial distances between immune cells and melanoma cells. In the current study, we aimed to validate BF multiplex IHC techniques in the Ventana Discovery Ultra Immunostainer to be applied to the evaluation of the TME in variably pigmented melanoma tissues. The BF multiplex IHC staining was performed using different combinations of six immune-cell markers—CD3, CD4, CD8, CD20, CD68 and CD163—and the melanoma cell marker SOX10. Our results show that the BF double IHC Yellow/Purple protocol guarantees the maximum contrast in all the cell populations tested and the combination SOX10 (Green), CD8 (Yellow) and CD163 (Purple) of the BF triple IHC protocol ensures the best contrast and discrimination between the three stained cell populations. Furthermore, the labeled cells were clearly distinct and easily identifiable using the image analysis software. Our standardized BF IHC multiplex protocols can be used to better assess the immune contexts of melanoma patients with potential applications to drive therapeutic decisions within clinical trials.

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“…Slides were airdried overnight, then placed in a vacuum sealed dehydrator for short-term storage. mIHC panels were optimized as according to Yaseen et al (27).…”

Section: Multiplex Immunohistochemistrymentioning

confidence: 99%

Detailed spatial immunophenotyping of primary melanomas reveals immune cell subpopulations associated with patient outcome

et al. 2022

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While the tumor immune microenvironment (TIME) of metastatic melanoma has been well characterized, the primary melanoma TIME is comparatively poorly understood. Additionally, although the association of tumor-infiltrating lymphocytes with primary melanoma patient outcome has been known for decades, it is not considered in the current AJCC melanoma staging system. Detailed immune phenotyping of advanced melanoma has revealed multiple immune biomarkers, including the presence of CD8+ T-cells, for predicting response to immunotherapies. However, in primary melanomas, immune biomarkers are lacking and CD8+ T-cells have yet to be extensively characterized. As recent studies combining immune features and clinicopathologic characteristics have created more accurate predictive models, this study sought to characterize the TIME of primary melanomas and identify predictors of patient outcome. We first phenotyped CD8+ T cells in fresh stage II primary melanomas using flow cytometry (n = 6), identifying a CD39+ tumor-resident CD8+ T-cell subset enriched for PD-1 expression. We then performed Opal multiplex immunohistochemistry and quantitative pathology-based immune profiling of CD8+ T-cell subsets, along with B cells, NK cells, Langerhans cells and Class I MHC expression in stage II primary melanoma specimens from patients with long-term follow-up (n = 66), comparing patients based on their recurrence status at 5 years after primary diagnosis. A CD39+CD103+PD-1- CD8+ T-cell population (P2) comprised a significantly higher proportion of intratumoral and stromal CD8+ T-cells in patients with recurrence-free survival (RFS) ≥5 years vs those with RFS <5 years (p = 0.013). Similarly, intratumoral B cells (p = 0.044) and a significantly higher B cell density at the tumor/stromal interface were associated with RFS. Both P2 and B cells localized in significantly closer proximity to melanoma cells in patients who remained recurrence-free (P2 p = 0.0139, B cell p = 0.0049). Our results highlight how characterizing the TIME in primary melanomas may provide new insights into how the complex interplay of the immune system and tumor can modify the disease outcomes. Furthermore, in the context of current clinical trials of adjuvant anti-PD-1 therapies in high-risk stage II primary melanoma, assessment of B cells and P2 could identify patients at risk of recurrence and aid in long-term treatment decisions at the point of primary melanoma diagnosis.

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Validation of an Accurate Automated Multiplex Immunofluorescence Method for Immuno-Profiling Melanoma

Cited by 15 publications

References 33 publications

Recent Advancement of PD-L1 Detection Technologies and Clinical Applications in the Era of Precision Cancer Therapy

Recent Advancement of PD-L1 Detection Technologies and Clinical Applications in the Era of Precision Cancer Therapy

Bright-Field Multiplex Immunohistochemistry Assay for Tumor Microenvironment Evaluation in Melanoma Tissues

Detailed spatial immunophenotyping of primary melanomas reveals immune cell subpopulations associated with patient outcome

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