Semi-Automated Image Analysis Methodology to Investigate Intracellular Heterogeneity in Immunohistochemical Stained Sections

The discovery of tissue heterogeneity revolutionized the existing knowledge regarding the cellular, molecular, and pathophysiological mechanisms in biomedicine. Therefore, basic science investigations were redirected to encompass observation at the classical and quantum biology levels. Various approaches have been developed to investigate and capture tissue heterogeneity; however, these approaches are costly and incompatible with all types of samples. In this paper, we propose an approach to quantify heterogeneous cellular populations through combining histology and images processing techniques. In this approach, images of immunohistochemically stained sections are processed through color binning of DAB-stained cells (in brown) and non-stained cells (in blue) to select cellular clusters expressing biomarkers of interest. Subsequently, the images were converted to a binary format through threshold modification (threshold 60%) in the grey scale. The cell count was extrapolated from the binary images using the particle analysis tool in ImageJ. This approach was applied to quantify the level of progesterone receptor expression levels in a breast cancer cell line sample. The results of the proposed approach were found to closely reflect those of manual counting. Through this approach, quantitative measures can be added to qualitative observation of subcellular targets expression

A novel image analysis approach to characterise the effects of dietary components on intestinal morphology and immune system in Atlantic salmon

The intestinal tract of salmonids provides a dynamic interface that not only mediates nutrient uptake but also functions as the first line of defence against ingested pathogens. Exposure of the immune system to beneficial microorganisms and different dietary immunostimulants via the intestine has been shown to prime the immune system and help in the development of immune competence. Furthermore, the morphology and function of teleostean intestines are known to respond to feed components and to ingested and resident bacterial communities. Histological appraisal is still generally considered to be the gold standard for sensitive assessment of the effects of such dietary modulation. The aim of the present study was to improve understanding of salmonid intestinal function, structure and dynamics and to use the knowledge gained to develop a model for analysis, which would allow intestinal health to be assessed with respect to different intestinal communities and feed components. Virtual histology, the process of assessing digital images of histological slides, is gaining momentum as an approach to supplement traditional histological evaluation methodologies and at the same time, image analysis of digitised histological sections provides a practical means for quantifiable assessment of structural and functional changes in tissues, being both objective and reproducible. This project focused on the development of a rapid, practical analytical methodology based on advanced image analysis, that was able to measure and characterise a range of features of the intestinal histology of Atlantic salmon in a quantitative manner. In the first research chapter, the development of a novel histological assessment system based upon advanced image analysis was described, this being developed with the help of a soybean feed model known to induce enteropathy in Atlantic salmon. This tool targeted the evaluation of the extent of morphological changes occurring in the distal intestine of Atlantic salmon following dietary modulation. The final analytical methodology arrived at, could be conducted with minimal user-interaction, allowing rapid and objective assessment of 12 continuous variables per histological frame analysed. The processing time required for each histological frame was roughly 20-25 min, which greatly improved the efficiency of conducting such a quantitative assessment with respect to the time taken for a subjective semi-quantitative alternative approach. Significant agreement between the fully automated and the manual morphometric image segmentation was achieved, however, the strength of this quantitative approach was enhanced by the employment of interactive procedures, which enabled the operator / observer to rectify preceding automated segmentation steps, and account for the specimen’s variations. Results indicated that image analysis provided a viable alternative to a pathologist’s manual scoring, being more practical and time-efficient. In the second research chapter, feeding Atlantic salmon a high inclusion level of unrefined SBM (25 %) produced an inflammatory response in the distal intestine as previously described by other authors. The model feed trial successfully generated differentiable states, although these were not, for the most part, systemically differentiable through the majority of standard immunological procedures used, being only detectable morphologically. Quantitation of morphometric parameters associated with histological sections using the newly developed image analysis tool successfully allowed identification of major morphological changes. Image analysis was thus shown to provide a powerful tool for describing the histomorphological structure of Atlantic salmon distal intestine. In turn, the semi-automated image analysis methods were able to distinguish normal intestinal mucosa from those affected by enteritis. While individual parameters were less discriminatory, use of multivariate techniques allowed better discrimination of states and is likely to prove the most productive approach in further studies. Work described in the third research chapter sought to validate the semi-automated image analysis system to establish that it was measuring the parameters it was purported to be measuring, and to provide reassurance that it could reliably measure pre-determined features. This study, using the same sections for semi-quantitative and quantitative analyses, demonstrated that the quantitative indices performed well when compared to analogous semi-quantitative descriptive parameters of assessment for enteritis prognosis. The excellent reproducibility and accuracy performance levels indicated that the image analysis system was a useful and reliable morphometric method for the quantification of SB-induced enteritis in salmon. Other characteristics such as rapidity, simplicity and adaptability favour this method for image analysis, and are particularly useful where less experienced interpreters are performing the analysis. The work described in the fourth research chapter characterised changes in the morphology of the intestinal epithelial cells occurring as a result of dietary modulation and aspects of inflammatory infiltration, using a selected panel of enzyme and IHC markers. To accomplish this, image analysis techniques were used to evaluate and systematically optimise a quantitative immunolabelling assessment protocol. Digital computer-assisted quantification of labelling for cell proliferation and regeneration; programmed cell death or apoptosis; EGCs and t-cell like infiltrates; mobilisation of stress-related protein regenerative processes and facilitation of nutrient uptake and ion transport provided encouraging results. Through the description of the intestinal cellular responses at a molecular level, such IHC expression profiling further characterised the inflammatory reaction generated by the enteropathic diet. In addition, a number of potential diagnostic parameters were described for fish intestinal health e.g. the relative levels of antigenicity and the spatial distribution of antigens in tissues. Work described in the final research chapter focused on detailed characterisation of intestinal MCs / EGCs in order to try to elucidate their functional role in the intestinal immune responses. Through an understanding of their distribution, composition and ultrastructure, the intention was to better characterise these cells and their functional properties. The general morphology, histochemical characteristics and tissue distribution of these cells were explored in detail using histochemical, IHC and immunogold staining / labelling, visualised using light, confocal and TEM microscopy. Despite these extensive investigations, their physiological function and the content of their granules still remain somewhat obscure, although a role as immunodulatory cells reacting to various exogeneous signals through a finely regulated process and comparable to that causing the degranulation of mammalian MCs is suggested. The histochemical staining properties demonstrated for salmonid MCs / EGCs seem to resemble those of mammalian mucosal mast cells, with both acidophilic and basophilic components in their granules, and a granule content containing neuromodulator / neurotransmitter-peptides such as serotonin, met-enkephalin and substance-p. Consequently, distinguishable bio-chromogenic markers have been identified that are of utility in generating a discriminatory profile for image analysis of such cells

Targeted computational analysis of the C3HEB/FEJ mouse model for drug efficacy testing

2020 Spring.Includes bibliographical references.Efforts to develop effective and safe drugs for the treatment of tuberculosis (TB) require preclinical evaluation in animal models. Alongside efficacy testing of novel therapies, effects on pulmonary pathology and disease progression are monitored by using histopathology images from these infected animals. To compare the severity of disease across treatment cohorts, pathologists have historically assigned a semi-quantitative histopathology score that may be subjective in terms of their training, experience, and personal bias. Manual histopathology, therefore, has limitations regarding reproducibility between studies and pathologists, potentially masking successful treatments. This report describes a pathologist-assistive software tool that reduces these user limitations while providing a rapid, quantitative scoring system for digital histopathology image analysis. The software, called 'Lesion Image Recognition and Analysis' (LIRA), employs convolutional neural networks to classify seven different pathology features, including three different lesion types from pulmonary tissues of the C3HeB/FeJ tuberculosis mouse model. LIRA was developed to improve the efficiency of histopathology analysis for mouse tuberculosis infection models. The model approach also has broader applications to other diseases and tissues. This also includes animals that are undergoing anti-mycobacterial treatment and host immune system modulation. A complimentary software package called 'Mycobacterial Image Analysis' (MIA) had also been developed that characterizes the varying bacilli characteristics such as density, aggregate/planktonic bacilli size, fluorescent intensity, and total counts. This further groups the bacilli characteristic data depending on the seven different classifications that are selected by the user. Using this approach allows for an even more targeted analysis approach that can determine how therapy and microenvironments influence the Mtb response

Role of angiogenic factors and circulating monocytes in the pathogenesis of preeclampsia and intrauterine fetal growth restriction

A collection of studies into the pathogenesis of preeclampsia (PE) and intrauterine fetal growth restriction (IUGR) are presented, focusing on factors involved in the angiogenesis as well as maternal factors such as circulating monocytes and lipid status. Placental expression of PlGF and KDR were significantly reduced in IUGR Adescription of digital image analysis techniques in the assessment of angiogenic factor expression in the placenta is presented. The study on circulating angiogenic factors showed elevated sFlt-1 and sEndoglin and low PlGF in preeclampsia and IUGR. Pro and anti-angiogenic factors and their ratios were assessed as biomarkers for pathological pregnancies. Maternal and fetal monocyte phenotype and polarization were examined inPE and IUGR. Ahigher percentage of intermediate and non-classical monocytes were found in PE and IUGR. Evaluation of inflammatory and healing monocyte phenotypes showed a shift towards healing phenotype in IUGR. The maternal and fetal triglyceride levels were higher in preeclampsia. This study documented the first description of elevated Apo lipoprotein B levels in cord blood at delivery in PE and IUGR. This research contributes to the literature on pathogenesis of preeclampsia and intrauterine growth restriction, demonstrating similarities and differences between the conditions which has lead us closer towards understanding their pathogenesis

Mycolactone-dependent depletion of endothelial cell thrombomodulin is strongly associated with fibrin deposition in Buruli ulcer lesions

A well-known histopathological feature of diseased skin in Buruli ulcer (BU) is coagulative necrosis caused by the Mycobacterium ulcerans macrolide exotoxin mycolactone. Since the underlying mechanism is not known, we have investigated the effect of mycolactone on endothelial cells, focussing on the expression of surface anticoagulant molecules involved in the protein C anticoagulant pathway. Congenital deficiencies in this natural anticoagulant pathway are known to induce thrombotic complications such as purpura fulimans and spontaneous necrosis. Mycolactone profoundly decreased thrombomodulin (TM) expression on the surface of human dermal microvascular endothelial cells (HDMVEC) at doses as low as 2ng/ml and as early as 8hrs after exposure. TM activates protein C by altering thrombin's substrate specificity, and exposure of HDMVEC to mycolactone for 24 hours resulted in an almost complete loss of the cells' ability to produce activated protein C. Loss of TM was shown to be due to a previously described mechanism involving mycolactone-dependent blockade of Sec61 translocation that results in proteasome-dependent degradation of newly synthesised ER-transiting proteins. Indeed, depletion from cells determined by live-cell imaging of cells stably expressing a recombinant TM-GFP fusion protein occurred at the known turnover rate. In order to determine the relevance of these findings to BU disease, immunohistochemistry of punch biopsies from 40 BU lesions (31 ulcers, nine plaques) was performed. TM abundance was profoundly reduced in the subcutis of 78% of biopsies. Furthermore, it was confirmed that fibrin deposition is a common feature of BU lesions, particularly in the necrotic areas. These findings indicate that there is decreased ability to control thrombin generation in BU skin. Mycolactone's effects on normal endothelial cell function, including its ability to activate the protein C anticoagulant pathway are strongly associated with this. Fibrin-driven tisischemia could contribute to the development of the tissue necrosis seen in BU lesions

Exploring biomarkers from the tumour and the microenvironment in Diffuse Large B-cell Lymphoma.

PhDIn the last decade unprecedented improvement in cure rates and overall survival was achieved in diffuse large B-cell Lymphoma (DLBCL) through the introduction of rituximab and anthracyclin-based chemotherapy (R-CHOP) as first line treatment. However, 40% of patients are refractory or relapse after R-CHOP and are hardly salvaged. To date, only age, International Prognostic Index (IPI) stratification and genetic aberrations defining gray-zone lymphomas have been used in clinical trials to select high-risk patients for more aggressive regimens. However, these prognostic features do not take into account the full biological heterogeneity of DLBCL. This reflects our limited knowledge on comprehensive prognostication in this group of disorders and supports our choice to investigate old and new prognostic factors for DLBCL in this thesis. Molecular characterization is generating opportunities for personalized therapy in poor-risk DLBCL. In order for targeted therapies to succeed in this disease, reliable and reproducible strategies that adequately segregate patients into distinct molecular groups are needed. While gene expression profiling (GEP) is the gold standard method, there is presently a lack of standardized methodology for array analysis, which can lead to variable results. The lack of a routine methodology for GEP has led investigators to develop immunohistochemistry (IHC) based approaches for the molecular classification in DLBCL. In fact, the Hans algorithm is being used to identify non-GCB DLBCLs in clinical trials offering NF-kB targeting agents to patients with this subtype. By performing a systematic comparison of nine IHC algorithms for molecular classification in a new large dataset of diagnostic DLBCL, we document an extremely low concordance across all classifiers (<21%) when classifying each individual patient, and a lack of outcome impact of all strategies, demonstrating that IHC is not a reliable alternative to molecular-based methods to be used for clinical decisions in DLBCL. GEP studies also suggested that the microenvironment could provide prognostic biomarkers in DLBCL in the R-CHOP era. Most authors have focused on the use of IHC to enumerate and functionally characterize the microenvironment in DLBCL. In our second study, by comparing two methods of semi-automated analysis for IHC staining Abstract 6 of the microenvironment, we demonstrate that the computerized results are highly reproducible, add the required robustness to IHC studies and should be used in the future instead of manual analysis. By applying comprehensive statistical analysis we propose that CD3 and FoxP3 should be validated as predictors of response to R-CHOP in clinical trials. Whereas a number of mechanisms by which cancer cells influence macrophage function have been described, currently there is very limited understanding of the macrophage polarisation status and effector function in human DLBCL. In our third study we analysed the GEP of macrophages sorted from human DLBCL samples. Unsupervised hierarchical clustering does not resolve DLBCL macrophage samples from reactive macrophage samples, indicating that macrophage heterogeneity in DLBCL should be considered. 202 genes are differentially expressed in DLBCL relative to controls. Functional annotation supports that these genes are macrophage-specific. We demonstrate that DLBCL macrophages have a bidirectional M1 and M2 functional activation, challenging the concept, widespread in the literature, that macrophages in tumours have a predominant M2 transcriptome. In our fifth study we used a two-cell co-culture model in an attempt to demonstrate that DLBCL cells influence macrophage transcriptome and proteome. The heterogeneity of the results, which precludes the confirmation of our hypothesis, is fully discussed. In our last study we tease out the DLBCL macrophage GEP heterogeneity and propose IFN- as a culprit B-cell derived molecule influencing macrophage activation status. Finally, using immunofluorescence we demonstrate that both M1 and M2 proteins are expressed in DLBCL macrophages.This work was supported by the portuguese institution Fundação para a Ciência e Tecnologia, grant number SFRH / BD / 68462 / 2010

A study of the molecular pathology of ductal carcinoma in situ and invasive ductal carcinoma of the breast.

The biological validity of the histopathological classification of ductal carcinoma in situ (DCIS) of the breast was evaluated in this study by correlating the three histopathological grades of DCIS to immunohistochemical expression of Ki67, p53, cerbB-2, markers of poor prognosis in invasive ductal carcinoma (IDC) and also to bcl2 and ER, markers of good prognosis in invasive breast cancer. DCIS grades correlated positively to Ki67, p53, cerbB-2 and negatively to bcl2 and ER, suggesting validity of the classification. The incidence of bax protein expression was determined immunohistochemically in DCIS and IDC. It did not correlate to histopathological grades of DCIS or IDC. The relationships of bax protein to the above mentioned biological markers were also determined in DCIS and IDC. Furthermore, the expression of bax, bcl2, Ki67, ER, p53 and cerbB-2 within DCIS grades was compared with the expression of these markers within IDC grades. The DCIS grades were determined subjectively as well as objectively by means of computer assisted image analysis with significant correlation found between subjective and objective measures. Image analysis was also used to determine percentage of positive cells per case for the nuclear stains (Ki67, ER, p53). Immunohistochemically positive p53 cases were analysed for p53 mutation by polymerase chain reaction (PCR) and subsequent DNA sequencing to compare the incidence of p53 mutation in DCIS to that of IDC. Biochemical changes within tissue may either initiate disease or occur as the result of the disease process and these changes can be studied by both Fourier transform infrared (FTIR) and FT-Raman spectroscopic techniques. FTIR and FT-Raman were employed to distinguish the DCIS and IDC grades. It has the potential to distinguish between DCIS grades, between IDC grades and also between DCIS and IDC as whole groups. The implications of the obtained data for the understanding of the molecular biology of DCIS of the breast and IDC are discussed and future investigations to further elucidate the molecular and cellular mechanisms involved are proposed

IDENTIFYING PREDICTIVE MARKERS OF RESPONSE AND RESISTANCE TO POTENT MULTI-AGENT HER2-TARGETED THERAPY

Background: Dual HER2 blockade with lapatinib plus trastuzumab without chemotherapy resulted in a substantial pathologic complete response (pCR) rate in HER2+ breast cancer patients (TBCRC006). Aberrant activation of the PI3K pathway as well as levels of tumor infiltrating lymphocytes (TILs) at diagnosis has been shown to be associated with response to HER2 targeted therapy. However, the results from these clinical trials are confounded by the co-administration of chemotherapy. In this study, we investigated the predictive value of PI3K-pathway activation and TILs in patients receiving neoadjuvant HER2 targeted therapy without chemotherapy. Methods: Pre-treatment biopsies from patients enrolled in the TBCRC006 trial were used for the aims of this study. PTEN expression status and PIK3CA mutations were assessed by immunohistochemistry and targeted sequencing of tumor DNA, respectively. Hematoxylin and eosin (H&E)-stained slides were evaluated for the % of stromal TILs; a threshold of 60% was used to define high TILs. Single formalin-fixed paraffin-embedded slides from 10 cases were co-stained for CD4, CD8, CD20, CD68, FoxP3, cytokeratin, and DAPI by multiplexed immunofluorescence (m-IF) using PerkinElmer Opal system. Multispectral imaging and digital analysis to visualize and quantify specific immune infiltrates were performed using PerkinElmerVectra system. The results were correlated with pathologic complete response (pCR). Results: Of the 64 evaluable patients, tumor tissue was available from 59 patients for PTEN immunohistochemistry, and DNA was available from 38 cases for mutation analysis. PTEN status (dichotomized by H-score median) was correlated with pCR (32% in high PTEN vs. 9% in low PTEN, p=0.04). PIK3CA mutations were identified in 12/38 tumors (32%) and were independent of ER or PTEN expression. No patient whose tumor harbored a PIK3CA mutation achieved pCR (p=0.07). When considered together (35 cases), 0/19 cases (0%) with a PIK3CA mutation and/or PTEN low expression had a pCR compared to 5/16 cases (31%) with PI3KCA wild type and high PTEN (p=0.01). TILs evaluation was available for 59 of the 64 patients who were evaluable for pathologic response. Twelve of 59 pts (20%) exhibited High TILs. The pCR rate was numerically higher in high-TILs compared to low-s-TILs (50% vs. 19%, P = 0.057). Multispectral imaging successfully captured and quantified multiple immune cell types in all the so far m-IF-stained samples. The density of stromal TILs (calculated as the total number of cells positive for the above immune markers/mm2) significantly correlated with % of stromal TILs evaluated on the H&E-stained slides (r = 0.76, P = 0.01). Conclusion: PI3K pathway hyperactivation is associated with resistance to lapatinib and trastuzumab without chemotherapy. If externally confirmed, future studies should investigate targeting PI3K/Akt/mTOR, in addition to HER2, in patients with HER2-positive tumors and evidence of aberrant downstream PI3K pathway activation. High levels of stromal TILs were associated with a numerically higher pCR rate than low stromal TILs in patients with HER2+ breast cancer treated with anti-HER2 agents without chemotherapy. However, the p-value did not reach statistical significance. Characterizing TILs with m-IF is feasible and may help correlate various TIL subpopulations with response to treatment