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ObjectivesThe interaction between the immune system and tumor cells is an important feature for the prognosis and treatment of cancer. Multiplex immunohistochemistry (mIHC) and multiplex immunofluorescence (mIF) analyses are emerging technologies that can be used to help quantify immune cell subsets, their functional state, and their spatial arrangement within the tumor microenvironment.MethodsThe Society for Immunotherapy of Cancer (SITC) convened a task force of pathologists and laboratory leaders from academic centers as well as experts from pharmaceutical and diagnostic companies to develop best practice guidelines for the optimization and validation of mIHC/mIF assays across platforms.ResultsRepresentative outputs and the advantages and disadvantages of mIHC/mIF approaches, such as multiplexed chromogenic IHC, multiplexed immunohistochemical consecutive staining on single slide, mIF (including multispectral approaches), tissue-based mass spectrometry, and digital spatial profiling are discussed.ConclusionsmIHC/mIF technologies are becoming standard tools for biomarker studies and are likely to enter routine clinical practice in the near future. Careful assay optimization and validation will help ensure outputs are robust and comparable across laboratories as well as potentially across mIHC/mIF platforms. Quantitative image analysis of mIHC/mIF output and data management considerations will be addressed in a complementary manuscript from this task force.

ObjectivesMultiplex immunohistochemistry and immunofluorescence (mIHC/IF) are emerging technologies that can be used to help define complex immunophenotypes in tissue, quantify immune cell subsets, and assess the spatial arrangement of marker expression. mIHC/IF assays require concerted efforts to optimize and validate the multiplex staining protocols prior to their application on slides. The best practice guidelines for staining and validation of mIHC/IF assays across platforms were previously published by this task force. The current effort represents a complementary manuscript for mIHC/IF analysis focused on the associated image analysis and data management.MethodsThe Society for Immunotherapy of Cancer convened a task force of pathologists and laboratory leaders from academic centers as well as experts from pharmaceutical and diagnostic companies to develop best practice guidelines for the quantitative image analysis of mIHC/IF output and data management considerations.ResultsBest-practice approaches for image acquisition, color deconvolution and spectral unmixing, tissue and cell segmentation, phenotyping, and algorithm verification are reviewed. Additional quality control (QC) measures such as batch-to-batch correction and QC for assembled images are also discussed. Recommendations for sharing raw outputs, processed results, key analysis programs and source code, and representative photomicrographs from mIHC/IF assays are included. Lastly, multi-institutional harmonization efforts are described.ConclusionsmIHC/IF technologies are maturing and are routinely included in research studies and moving towards clinical use. Guidelines for how to perform and standardize image analysis on mIHC/IF-stained slides will likely contribute to more comparable results across laboratories and pave the way for clinical implementation. A checklist encompassing these two-part guidelines for the generation of robust data from quantitative mIHC/IF assays will be provided in a third publication from this task force. While the current effort is mainly focused on best practices for characterizing the tumor microenvironment, these principles are broadly applicable to any mIHC/IF assay and associated image analysis.

Prolactin (PRL) and growth hormone (GH) belong to a family of closely related hormones, which bind two receptors in a sequential order on two distinct faces of the ligand (sites I and II). Phosphorylated forms of both PRL and GH have been described in numerous species. The functional significance of these hormone variants is uncertain. Phosphorylation of bovine PRL (bPRL) in the pituitary occurs mostly at the designated major site serine 90 (S90) and results in marked loss of biological activity of the modified hormone. This dissertation examines the effect of a mimic of phosphorylation of bovine GH (bGH) at the residue equivalent to S90 in bPRL (S84) using a transgenic mouse model. Mimicry of phosphorylation was accomplished by substituting S84 with glutamic acid in the transgene. Overexpression of the mutant hormone failed to induce any of the characteristic features of the phenotype of GH transgenic mice. Abnormalities in growth, organ lesions, or markedly reduced fertility could not be detected in the transgenic model. In a second in vitro study we examined the effect of substitution at S90 in bPRL with polar, charged, or hydrophobic amino acids to elucidate which of the physical and chemical properties of phosphorylated S90 are important for this mechanism of phosphorylation-induced loss in biological activity. Substitution of S90 in bPRL with various amino acids demonstrated that size and polarity were especially important. Specific amino acids surrounding serine 90 which form a salt bridge were also mutated and were demonstrated to be involved as well. Mutations at S90 were also associated with a marked right-shift in the agonist phase of the dose-response curve, while the antagonist phase remained unchanged. These results indicate that mutation at his site primarily disrupts the function of site II. In a final in vitro study we compared the effect of phosphorylation at identified sites in rat, human, and bovine PRL by mutational analyses using recombinant hormones. A common pattern of the mutation-induced changes could not be identified demonstrating distinct differences between species.