Identification of distinct immune landscapes using an automated nine-color multiplex immunofluorescence staining panel and image analysis in paraffin tumor tissues

Our plain-English summary of Identification of distinct immune landscapes using an automated nine-color multiplex immunofluorescence staining panel and image analysis in paraffin tumor tissues, by E.R. Parra, J. Zhai, A. Tamegnon, et al (Scientific Reports, 2021).

The study

• Authors: E.R. Parra, J. Zhai, A. Tamegnon, N. Zhou, R.K. Pandurengan, C. Barreto, M. Jiang, D.C. Rice, C. Creasy, A.A. Vaporciyan, W.L. Hofstetter, A.S. Tsao, I.I. Wistuba, B. Sepesi, C. Haymaker
• Journal: Scientific Reports
• Published: 2021;11(1):4530
• Cited by: 42 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Immune profiling is becoming a vital tool for identifying predictive and prognostic markers for translational studies. The study of the tumor microenvironment (TME) in paraffin tumor tissues such as malignant pleural mesothelioma (MPM) could yield insights to actionable targets to improve patient outcome. Here, we optimized and tested a new immune-profiling method to characterize immune cell phenotypes in paraffin tissues and explore the co-localization and spatial distribution between the immune cells within the TME and the stromal or tumor compartments. Tonsil tissues and tissue microarray (TMA) were used to optimize an automated nine-color multiplex immunofluorescence (mIF) panel to study the TME using eight antibodies: PD-L1, PD-1, CD3, CD8, Foxp3, CD68, KI67, and pancytokeratin. To explore the potential role of the cells into the TME with this mIF panel we applied this panel in twelve MPM cases to assess the multiple cell phenotypes obtained from the image analysis and well as their spatial distribution in this cohort. We successful optimized and applied an automated nine-color mIF panel to explore a small set of MPM cases. Image analysis showed a high degree of cell phenotype diversity with immunosuppression patterns in the TME of the MPM cases. Mapping the geographic cell phenotype distribution in the TME, we were able to identify two distinct, complex immune landscapes characterized by specific patterns of cellular distribution as well as cell phenotype interactions with malignant cells. Successful we showed the optimization and reproducibility of our mIF panel and their incorporation for comprehensive TME immune profiling into translational studies that could refine our ability to correlate immunologic phenotypes with specific patterns of cells distribution and distance analysis. Overall, this will improve our ability to understand the behavior of cells within the TME and predict new treatment strategies to improve patient outcome.

In plain English

This was a laboratory methods study. The authors set up an automated technique that stains eight proteins at once on paraffin tissue slides (nine colors, including a nuclear stain), so that image analysis can identify immune cells and cancer cells and measure the distances between them. They developed it on tonsil tissue, then tried it on tumor samples from 12 patients with malignant pleural mesothelioma, randomly selected from the MD Anderson Cancer Center tissue bank. Asbestos exposure was among the clinical details collected, but the article's text reports no results by asbestos exposure. Staining repeated in three consecutive weeks showed positive and significant correlations. All 12 tumors were classified PD-L1 positive (more than 1% of cancer cells). Cytotoxic T cells were the dominant T-cell type, at a median of 57.67 cells per square millimetre. Exploratory comparisons found significantly higher densities of some T-cell types in tumors from ever-smokers than from never-smokers. T cells and cancer cells showed a mixed pattern in 7 of 12 cases and an unmixed pattern in 5. The authors concluded the method was reproducible. They described the study as small, descriptive and exploratory, and said it needs validation in a larger group.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory methods development study (multiplex immunofluorescence staining and image analysis), with an exploratory application to a small set of tumor samples
• Who was studied: Tissue from 12 malignant pleural mesothelioma cases (11 epithelioid, 1 biphasic), randomly selected from the authors' archived tissue bank at MD Anderson Cancer Center, Houston. All patients had received platinum and pemetrexed chemotherapy before surgery. Tonsil tissue was used to develop the staining. Collection dates are not given.
• Exposure: Not an exposure study. Asbestos exposure was among the clinical details retrieved from patient records (in a supplementary table not included in the text), but the text reports no analysis or results by asbestos exposure. Fiber types are not mentioned.
• Main results:
  • Staining the tissue microarray three times, one week apart, gave positive and significant correlations between each pair of weeks.
  • All 12 mesothelioma cases were classified PD-L1 positive, using a cutoff of more than 1% of malignant cells expressing PD-L1.
  • The dominant T-cell type in the 12 cases (both tumor compartments combined) was cytotoxic T cells (CD3+CD8+): median 57.67 cells/mm2, minimum 26.23, maximum 175.35.
  • In this small exploratory comparison, tumors from ever-smokers had significantly higher densities of three T-cell types than tumors from never-smokers (P = 0.048, P = 0.018 and P = 0.018).
  • Two spatial patterns of T cells and cancer cells were identified. A 'mixed' pattern (close interaction) was seen in 7 of 12 cases and an 'unmixed' pattern (low interaction) in 5 of 12 cases.
  • Cytotoxic T cells overall showed an unmixed pattern, which the authors read as suggesting poor or weak interaction with malignant cells.
  • Cytotoxic T cells and antigen-experienced (PD-1 positive) T cells were closer to PD-L1-positive malignant cells than to PD-L1-negative malignant cells.
• The authors' conclusion: The authors concluded that they had shown the method is reproducible and can be used for immune profiling in translational studies. They said future validation in a larger group of patients is needed to confirm the correlations seen.
• Limitations the authors state:
  • Small number of mesothelioma cases.
  • The study is largely descriptive and exploratory, and its findings need to be extended.

Topics

Medical terms in this study, explained in our Lexicon: Tumor Microenvironment, Pleural Mesothelioma, PD-L1.

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