Standardized Processing for Formalin-Fixed, Paraffin-Embedded Cell Pellet Immunohistochemistry Controls.

Positive and negative controls with known expression of target proteins are essential for the development of immunohistochemistry (IHC) assays. While tissue controls are beneficial for well-characterized proteins with defined tissue and cellular expression patterns, they are less suitable for the initial development of IHC assays for novel, poorly characterized, or ubiquitously expressed proteins. Alternatively, due to their standardized nature, cell pellets, including cancer cell lines with defined protein or transcript expression levels (e.g., high, medium, and low expression), transfected over-expressing cell lines, or cell lines with genes deleted through cell engineering technologies like CRISPR, can serve as valuable controls, especially for the initial antibody characterization and selection. In order for these cell pellets to be used in the development of IHC assays for formalin-fixed, paraffin-embedded tissues, they need to be processed and embedded in a manner that recapitulates the procedures used for tissue processing. This protocol describes a process for creating and processing formalin-fixed, paraffin-embedded cell pellet controls that can be used for IHC method developments.

Synthetic Antigen Controls for Immunohistochemistry.

Immunohistochemistry (IHC) assays provide valuable insights into protein expression patterns, the reliable interpretation of which requires well-characterized positive and negative control samples. Because appropriate tissue or cell line controls are not always available, a simple method to create synthetic IHC controls may be beneficial. Such a method is described here. It is adaptable to various antigen types, including proteins, peptides, or oligonucleotides, in a wide range of concentrations. This protocol explains the steps necessary to create synthetic antigen controls, using as an example a peptide from the human erythroblastic oncogene B2 (ERBB2/HER2) intracellular domain (ICD) recognized by a variety of diagnostically relevant antibodies. Serial dilutions of the HER2 ICD peptide in bovine serum albumin (BSA) solution are mixed with formaldehyde and heated for 10 min at 85 °C to solidify and cross-link the peptide/BSA mixture. The resulting gel can be processed, sectioned, and stained like a tissue, yielding a series of samples of known antigen concentrations spanning a wide range of staining intensities. This simple protocol is consistent with routine histology lab procedures. The method requires only that the user have a sufficient quantity of the desired antigen. Recombinant proteins, protein domains, or linear peptides that encode relevant epitopes may be synthesized locally or commercially. Laboratories generating in-house antibodies can reserve aliquots of the immunizing antigen as the synthetic control target. The opportunity to create well-defined positive controls across a wide range of concentrations allows users to assess intra- and inter-laboratory assay performance, gain insight into the dynamic range and linearity of their assays, and optimize assay conditions for their particular experimental goals.

Characterization of Tumor-immune Microenvironment by High-throughput Image Analysis of CD8 Immunohistochemistry Combined With Modified Masson's Trichrome.

With the advent of checkpoint inhibitors, there is increasing need to study the dynamics of CD8+ T-cells in the tumor microenviroment. In this article, we describe a semi-automated method to quantify and interrogate spatial relationships between T-cells and collagenous stroma in human and mouse tissue samples. The assay combines CD8 immunohistochemistry with modified Masson's trichrome. Slides are scanned and digital images are analyzed using an adjustable MATLAB algorithm, allowing for high-throughput quantification of cytotoxic T-cells and collagen. This method provides a flexible tool for unbiased quantification of T-cells and their interactions with tumor cells and tumor microenvironment in tissue samples.

Integration of eQTL and a Single-Cell Atlas in the Human Eye Identifies Causal Genes for Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) is a leading cause of vision loss. To better understand disease pathogenesis and identify causal genes in GWAS loci for AMD risk, we present a comprehensive database of human retina and retinal pigment epithelium (RPE). Our database comprises macular and non-macular RNA sequencing (RNA-seq) profiles from 129 donors, a genome-wide expression quantitative trait loci (eQTL) dataset that includes macula-specific retina and RPE/choroid, and single-nucleus RNA-seq (NucSeq) from human retina and RPE with subtype resolution from more than 100,000 cells. Using NucSeq, we find enriched expression of AMD candidate genes in RPE cells. We identify 15 putative causal genes for AMD on the basis of co-localization of genetic association signals for AMD risk and eye eQTL, including the genes TSPAN10 and TRPM1. These results demonstrate the value of our human eye database for elucidating genetic pathways and potential therapeutic targets for ocular diseases.

Prognostic and predictive significance of proliferation in 867 colorectal cancers.

AIM: Recently, the Oncotype DX recurrence score, which measures a gene expression signature including markers of tumour proliferation, was validated as a prognostic signature in colorectal cancer. This study aimed to evaluate whether the Ki67 proliferation index can provide similar prognostic and predictive information. METHODS: Tissue microarrays were constructed from triplicate cores of colorectal cancer. Immunohistochemistry for Ki67 was performed with the SP6 antibody and the percentage of positive tumour cells scored. Prognostic significance was evaluated in 867 cancers (601 events) using Cox proportional hazards models. RESULTS: The Ki67 labelling index, divided at the median, was not a statistically or clinically significant prognostic factor in univariate analyses of 5-year overall survival (HR 0.98, 95% CI 0.84 to 1.15, p=0.84). Multivariate analyses were similarly non-significant. However, in Dukes' stage C patients, the high Ki67 subgroup derived a greater 5-year overall survival benefit from chemotherapy (HR 0.32, 95% CI 0.21 to 0.51, p<0.0001) than the low subgroup (HR 0.57, 95% CI 0.37 to 0.89, p=0.011). CONCLUSIONS: The Ki67 proliferation index is not a useful prognostic factor in colorectal cancer, but deserves further evaluation as a predictive factor for the incremental benefit derived from adjuvant chemotherapy.