Computational Immunohistochemistry: Recipes for Standardization of Immunostaining

Cancer diagnosis and personalized cancer treatment are heavily based on the visual assessment of immunohistochemically-stained tissue specimens. The precision of this assessment depends critically on the quality of immunostaining, which is governed by a number of parameters used in the staining process. Tuning of the staining-process parameters is mostly based on pathologists' qualitative assessment, which incurs inter- and intra-observer variability. The lack of standardization in staining across pathology labs leads to poor reproducibility and consequently to uncertainty in diagnosis and treatment selection. In this paper, we propose a methodology to address this issue through a quantitative evaluation of the staining quality by using visual computing and machine learning techniques on immunohistochemically-stained tissue images. This enables a statistical analysis of the sensitivity of the staining quality to the process parameters and thereby provides an optimal operating range for obtaining high-quality immunostains. We evaluate the proposed methodology on HER2-stained breast cancer tissues and demonstrate its use to define guidelines to optimize and standardize immunostaining

Antigen-antibody binding kinetics for quantitative molecular diagnostics

The binding of antibody and antigen constitutes the basis of immunoassays. In this thesis, we aim to explore this binding through three perspectives: a) improving the kinetics of the reaction by increasing mass transport, b) developing a novel method to study the kinetics of binding, and c) using immunoassays for quantifying surface antigen on tumors to uncover tumor heterogeneity. Nowadays, immunoassays are mostly performed on surfaces, which facilitates washing steps and exchanging reagents. A key limitation of such assays is the formation of a layer depleted of analyte, which reduces the reaction rate and thus decreases the overall sensitivity of the test. In the first part of this thesis, we explore the most common current strategies to reduce the depletion layer, i.e., the use of shakers. We show that while shakers are easy to use, they often offer unpredictable results. Such results can be greatly improved through the use of microfluidics, which offers the possibility to control mass transport through convective flows, improving signal while reducing total assay time. Nevertheless, microfluidics has not been adapted to the most common substrates used for immunoassays, microtiter plates. Thus, we propose a new microfluidic concept for controlling the mass transport in microtiter plate wells, which through the use of different kinetic zones allows the exploration of samples with high dynamic ranges in a single test. We then develop a method to evaluate the kinetics of antibody binding using fluorescence lifetime imaging microscopy. Such a method allows a characterization of kinetic constants by comparing the fraction of bound to unbound antibodies on a variety of substrates. We demonstrate its use in breast cancer derived cell blocks and in cancer tissues. Finally, we explore the heterogeneity in cancer, i.e., the different phenotypical profiles that cells in cancer show. Studying tumor heterogeneity is critical to understand cancer evolution in a patient and thus be able to perform accurate patient stratification. We develop a method easily implementable in pathology labs that uses the kinetics of binding of antibodies through immunohistochemical stains to quantify the presence of antigens on different regions of a sample. By performing this localized quantification, we can also explore the heterogeneity present on the tissue. We further investigate heterogeneity using a workflow involving the local tissue lysis and antibody microarray analysis. This method shows a higher capacity of multiplexing, which allowed us to investigate the relative variations of 13 proteins and their inter and intra-tumoral heterogeneity, highlighting the presence of multiple phenotypical variants in a single patient. Thus, in this thesis, we show several use scenarios of antibody-antigen binding kinetics, which highlight the potential of this simple binding to provide us with powerful techniques

Tissue lithography: Microscale dewaxing to enable retrospective studies on formalin-fixed paraffin-embedded (FFPE) tissue sections

We present a new concept, termed tissue lithography (TL), and its implementation which enables retrospective studies on formalin-fixed paraffin-embedded tissue sections. Tissue lithography uses a microfluidic probe to remove microscale areas of the paraffin layer on formalin-fixed paraffin-embedded biopsy samples. Current practices in sample utilization for research and diagnostics require complete deparaffinization of the sample prior to molecular testing. This imposes strong limitations in terms of the number of tests as well as the time when they can be performed on a single sample. Microscale dewaxing lifts these constraints by permitting deprotection of a fraction of a tissue for testing while keeping the remaining of the sample intact for future analysis. After testing, the sample can be sent back to storage instead of being discarded, as is done in standard workflows. We achieve this microscale dewaxing by hydrodynamically confining nanoliter volumes of xylene on top of the sample with a probe head. We demonstrate micrometer-scale, chromogenic and fluorescence-based immunohistochemistry against multiple biomarkers (p53, CD45, HER2 and β-actin) on tonsil and breast tissue sections and microarrays. We achieve stain patterns as small as 100 μm × 50 μm as well as multiplexed immunostaining within a single tissue microarray core with a 20-fold time reduction for local dewaxing as compared to standard protocols. We also demonstrate a 10-fold reduction in the rehydration time, leading to lower processing times between different stains. We further show the potential of TL for retrospective studies by sequentially dewaxing and staining four individual cores within the same tissue microarray over four consecutive days. By combining tissue lithography with the concept of micro-immunohistochemistry, we implement each step of the IHC protocol-dewaxing, rehydration and staining-with the same microfluidic probe head. Tissue lithography brings a new level of versatility and flexibility in sample processing and budgeting in biobanks, which may alleviate current sample limitations for retrospective studies in biomarker discovery and drug screening

Spatially multiplexed RNA in situ hybridization to reveal tumor heterogeneity

Multiplexed RNA in situ hybridization for the analysis of gene expression patterns plays an important role in investigating development and disease. Here, we present a method for multiplexed RNA-ISH to detect spatial tumor heterogeneity in tissue sections. We made use of a microfluidic chip to deliver ISH-probes locally to regions of a few hundred micrometers over time periods of tens of minutes. This spatial multiplexing method can be combined with ISH-approaches based on signal amplification, with bright field detection and with the commonly used format of formalin-fixed paraffin-embedded tissue sections. By using this method, we analyzed the expression of HER2 with internal positive and negative controls (ActB, dapB) as well as predictive biomarker panels (ER, PgR, HER2) in a spatially multiplexed manner on single mammary carcinoma sections. We further demonstrated the applicability of the technique for subtype differentiation in breast cancer. Local analysis of HER2 revealed medium to high spatial heterogeneity of gene expression (Cohen effect size r = 0.4) in equivocally tested tumor tissues. Thereby, we exemplify the importance of using such a complementary approach for the analysis of spatial heterogeneity, in particular for equivocally tested tumor samples. As the method is compatible with a range of ISH approaches and tissue samples, it has the potential to find broad applicability in the context of molecular analysis of human diseases

Spatial protein heterogeneity analysis in frozen tissues to evaluate tumor heterogeneity

A new workflow for protein-based tumor heterogeneity probing in tissues is here presented. Tumor heterogeneity is believed to be key for therapy failure and differences in prognosis in cancer patients. Comprehending tumor heterogeneity, especially at the protein level, is critical for tracking tumor evolution, and showing the presence of different phenotypical variants and their location with respect to tissue architecture. Although a variety of techniques is available for quantifying protein expression, the heterogeneity observed in the tissue is rarely addressed. The proposed method is validated in breast cancer fresh-frozen tissues derived from five patients. Protein expression is quantified on the tissue regions of interest (ROI) with a resolution of up to 100 μm in diameter. High heterogeneity values across the analyzed patients in proteins such as cytokeratin 7, β-actin and epidermal growth factor receptor (EGFR) using a Shannon entropy analysis are observed. Additionally, ROIs are clustered according to their expression levels, showing their location in the tissue section, and highlighting that similar phenotypical variants are not always located in neighboring regions. Interestingly, a patient with a phenotype related to increased aggressiveness of the tumor presents a unique protein expression pattern. In summary, a workflow for the localized extraction and protein analysis of regions of interest from frozen tissues, enabling the evaluation of tumor heterogeneity at the protein level is presented

Quantifying Antibody Binding Kinetics on Fixed Cells and Tissues via Fluorescence Lifetime Imaging

We present a method for monitoring spatially localized antigen–antibody binding events on physiologically relevant substrates (cell and tissue sections) using fluorescence lifetime imaging. Specifically, we use the difference between the fluorescence decay times of fluorescently tagged antibodies in free solution and in the bound state to track the bound fraction over time and hence deduce the binding kinetics. We make use of a microfluidic probe format to minimize the mass transport effects and localize the analysis to specific regions of interest on the biological substrates. This enables measurement of binding constants (kon) on surface-bound antigens and on cell blocks using model biomarkers. Finally, we directly measure p53 kinetics with differential biomarker expression in ovarian cancer tissue sections, observing that the degree of expression corresponds to the changes in kon, with values of 3.27–3.50 × 10(3) M(–1) s(–1) for high biomarker expression and 2.27–2.79 × 10(3) M(–1) s(–1) for low biomarker expression.ISSN:1520-6882ISSN:0003-270

Quantitative microimmunohistochemistry for the grading of immunostains on tumour tissues

Immunohistochemistry is the gold-standard method for cancer-biomarker identification and patient stratification. Yet, owing to signal saturation, its use as a quantitative assay is limited as it cannot distinguish tumours with similar biomarker-expression levels. Here, we introduce a quantitative microimmunochemistry assay that enables the acquisition of dynamic information, via a metric of the evolution of the immunohistochemistry signal during tissue staining, for the quantification of relative antigen density on tissue surfaces. We used the assay to stratify 30 patient-derived breast-cancer samples into conventional classes and to determine the proximity of each sample to the other classes. We also show that the assay enables the quantification of multiple biomarkers (human epidermal growth factor receptor, oestrogen receptor and progesterone receptor) in a standard breast-cancer panel. The integration of quantitative microimmunohistochemistry into current pathology workflows may lead to improvements in the precision of biomarker quantification

High-Quality Immunohistochemical Stains through Computational Assay Parameter Optimization

ISSN:0018-9294ISSN:1558-253