Project description:Multiplex immunofluorescence staining enables the simultaneous detection of multiple immune markers in a single tissue section, and is a useful tool for the identification of specific cell populations within the tumour microenvironment. However, this technology has rarely been validated against standard clinical immunohistology, which is a barrier for its integration into clinical practice. This study sought to validate and investigate the accuracy, precision and reproducibility of a multiplex immunofluorescence compared with immunohistochemistry (IHC), including tissue staining, imaging and analysis, in characterising the expression of immune and melanoma markers in both the tumour and its microenvironment. Traditional chromogenic IHC, single-plex immunofluorescence and multiplex immunofluorescence were each performed on serial tissue sections of a formalin-fixed paraffin-embedded (FFPE) tissue microarray containing metastatic melanoma specimens from 67 patients. The panel included the immune cell markers CD8, CD68, CD16, the immune checkpoint PD-L1, and melanoma tumour marker SOX10. Slides were stained with the Opal™ 7 colour Kit (Akoya Biosciences) on the intelliPATH autostainer (Biocare Medical) and imaged using the Vectra 3.0.5 microscope. Marker expression was quantified using Halo v.3.2.181 (Indica Labs). Comparison of the IHC and single-plex immunofluorescence revealed highly significant positive correlations between the cell densities of CD8, CD68, CD16, PD-L1 and SOX10 marker positive cells (Spearman's rho = 0.927 to 0.750, p < 0.0001). Highly significant correlations were also observed for all markers between single-plex immunofluorescence and multiplex immunofluorescence staining (Spearman's rho >0.9, p < 0.0001). Finally, correlation analysis of the three multiplex replicates revealed a high degree of reproducibility between slides (Spearman's rho >0.940, p < 0.0001). Together, these data highlight the reliability and validity of multiplex immunofluorescence in accurately profiling the tumour and its associated microenvironment using FFPE metastatic melanoma specimens. This validated multiplex panel can be utilised for research evaluating melanoma and its microenvironment, such as studies performed to predict patient response or resistance to immunotherapies.

Project description:Neurodegenerative diseases of the central nervous system are characterised by pathogenetic cellular and molecular changes in specific areas of the brain that lead to the dysfunction and/or loss of explicit neuronal populations. Despite exhibiting different clinical profiles and selective neuronal loss, common features such as abnormal protein deposition, dysfunctional cellular transport, mitochondrial deficits, glutamate excitotoxicity and inflammation are observed in most, if not all, neurodegenerative disorders, suggesting converging pathways of neurodegeneration. We have generated comparative genome-wide gene expression data for Alzheimer’s disease, amyotrophic lateral sclerosis, Huntington’s disease, multiple sclerosis, Parkinson’s disease and schizophrenia using an extensive cohort of well characterised post-mortem CNS tissues. The analysis of whole genome expression patterns across these major disorders offers an outstanding opportunity not only to look into exclusive disease specific changes, but more importantly to uncover potential common molecular pathogenic mechanisms that could be targeted for therapeutic gain. Surprisingly, no dysregulated gene that passed our selection criteria was found in common across all 6 diseases using our primary method of analysis. However, 61 dysregulated genes were shared when comparing five and four diseases. Our analysis indicates firstly the involvement of common neuronal homeostatic, survival and synaptic plasticity pathways. Secondly, we report changes to immunoregulatory and immunomodulatory pathways in all diseases. Our secondary method of analysis confirmed significant up-regulation of a number of genes in diseases presenting degeneration and showed that somatostatin was downregulated in all 6 diseases. The latter is supportive of a general role for neuroinflammation in the pathogenesis and/or response to neurodegeneration. Unravelling the detailed nature of the molecular changes regulating inflammation in the CNS is key to the development of novel therapeutic approaches for these chronic conditions. A total of 113 cases were selected retrospectively on the basis of a confirmed clinical and neuropathological diagnosis and snap-frozen brain blocks were provided by various tissue banks within the BrainNet Europe network. Total RNA was extracted from dissected snap-frozen tissue (< 100 mg) by the individual laboratories according to a BNE optimised common protocol using the RNeasy(r) tissue lipid mini kit (Qiagen Ltd, Crawley, UK) according to the manufacturer's instructions, and was stored at -80C until further use. Gene expression analysis was performed on the RNA samples using the Illumina whole genome HumanRef8 v2 BeadChip (Illumina, London, UK). All the labelling and hybridisation of the samples was carried out in a single experiment by the Imperial College group to reduce the technical variability. RNA samples were prepared for array analysis using the Illumina TotalPrep(tm)-96 RNA Amplification Kit (Ambion/Applied Biosystems, Warrington, UK). Finally, the BeadChips we re scanned using the Illumina BeadArray Reader. The data was extracted using BeadStudio 3.2 (Illumina). Data normalisation and gene differential expression analyses were conducted using the Rosetta error models available in the Rosetta Resolver(r) system (Rosetta Biosoftware, Seattle, Wa, USA). Two samples presented very low signal expression most likely due to hybridization problems and did not pass the quality control test. They are not represented here. One of the 2 samples was a replicate, therefore there was loss of only 1 case bringing the grand total of cases used to 112 (total of samples of 118 including 6 replicates).

Project description:Neurodegenerative diseases of the central nervous system are characterised by pathogenetic cellular and molecular changes in specific areas of the brain that lead to the dysfunction and/or loss of explicit neuronal populations. Despite exhibiting different clinical profiles and selective neuronal loss, common features such as abnormal protein deposition, dysfunctional cellular transport, mitochondrial deficits, glutamate excitotoxicity and inflammation are observed in most, if not all, neurodegenerative disorders, suggesting converging pathways of neurodegeneration. We have generated comparative genome-wide gene expression data for Alzheimer’s disease, amyotrophic lateral sclerosis, Huntington’s disease, multiple sclerosis, Parkinson’s disease and schizophrenia using an extensive cohort of well characterised post-mortem CNS tissues. The analysis of whole genome expression patterns across these major disorders offers an outstanding opportunity not only to look into exclusive disease specific changes, but more importantly to uncover potential common molecular pathogenic mechanisms that could be targeted for therapeutic gain. Surprisingly, no dysregulated gene that passed our selection criteria was found in common across all 6 diseases using our primary method of analysis. However, 61 dysregulated genes were shared when comparing five and four diseases. Our analysis indicates firstly the involvement of common neuronal homeostatic, survival and synaptic plasticity pathways. Secondly, we report changes to immunoregulatory and immunomodulatory pathways in all diseases. Our secondary method of analysis confirmed significant up-regulation of a number of genes in diseases presenting degeneration and showed that somatostatin was downregulated in all 6 diseases. The latter is supportive of a general role for neuroinflammation in the pathogenesis and/or response to neurodegeneration. Unravelling the detailed nature of the molecular changes regulating inflammation in the CNS is key to the development of novel therapeutic approaches for these chronic conditions.

Dataset Information

A manual multiplex immunofluorescence method for investigating neurodegenerative diseases.

Background

New method

Results

Comparison with existing methods

Conclusions

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