Improvements in digital pathology equipment for renal biopsies: updating the standard model.

INTRODUCTION: Digital pathology can improve the technical and interpretative workflows in nephropathology by creating hub-spoke networks and virtuous collaboration projects among centers in different geographical regions. New high-resolution fast-scanning instruments combined with currently existing equipment were tested in a nephropathology hub to evaluate possible upgrading in the routine processing phases. METHODS: The scanning performance of two different instruments (Aperio vs hybrid MIDI II) was evaluated and a comparative quality control check was performed on obtained whole slide images. RESULTS: Both with default and custom settings for light microscopy, MIDI II proved to be faster, with only slightly more time required to prepare the scan and larger final file size as compared to Aperio (p < 0.001). No differences were noted in the number of out-of-focus slides per case (p = 0.75). Regarding immunofluorescence, the new scanner required longer preparation time (p = 0.001) with comparable scanning times and final file size (p = 0.169 and p = 0.177, respectively). Quality control showed differences in 3 quality features related to white background and blurriness (p < 0.001). No major discordances in the final diagnosis were recorded after comparing the report obtained with slides scanned using the two instruments, with only one case (4%) showing minor disagreement. CONCLUSION: The present report describes the experience of a hub nephropathology center adopting next generation digital pathology tools for the routine assessment of renal biopsies, highlighting the need for a complementary approach towards a philosophy of interoperability.

Congo Red Staining in Digital Pathology: The Streamlined Pipeline for Amyloid Detection Through Congo Red Fluorescence Digital Analysis.

Renal amyloidosis is a rare condition caused by the progressive accumulation of misfolded proteins within glomeruli, vessels, and interstitium, causing functional decline and requiring prompt treatment due to its significant morbidity and mortality. Congo red (CR) stain on renal biopsy samples is the gold standard for diagnosis, but the need for polarized light is limiting the digitization of this nephropathology field. This study explores the feasibility and reliability of CR fluorescence on virtual slides (CRFvs) in evaluating the diagnostic accuracy and proposing an automated digital pipeline for its assessment. Whole-slide images from 154 renal biopsies with CR were scanned through a Texas red fluorescence filter (NanoZoomer S60, Hamamatsu) at the digital Nephropathology Center of the Istituto di Ricovero e Cura a Carattere Scientifico San Gerardo, Monza, Italy, and evaluated double-blinded for the detection and quantification through the amyloid score and a custom ImageJ pipeline was built to automatically detect amyloid-containing regions. Interobserver agreement for CRFvs was optimal (k = 0.90; 95% CI, 0.81-0.98), with even better concordance when consensus-based CRFvs evaluation was compared to the standard CR birefringence (BR) (k = 0.98; 95% CI, 0.93-1). Excellent performance was achieved in the assessment of amyloid score overall by CRFvs (weighted k = 0.70; 95% CI, 0.08-1), especially within the interstitium (weighted k = 0.60; 95% CI, 0.35-0.84), overcoming the misinterpretation of interstitial and capsular collagen BR. The application of an automated digital pathology pipeline (Streamlined Pipeline for Amyloid detection through CR fluorescence Digital Analysis, SPADA) further increased the performance of pathologists, leading to a complete concordance with the standard BR. This study represents an initial step in the validation of CRFvs, demonstrating its general reliability in a digital nephropathology center. The computational method used in this study has the potential to facilitate the integration of spatial omics and artificial intelligence tools for the diagnosis of amyloidosis, streamlining its detection process.

A DNA damage response-like phenotype defines a third of colon cancers at onset.

Colon adenocarcinoma (COAD) has a limited range of diversified, personalized therapeutic opportunities, besides DNA hypermutating cases; thus, both new targets or broadening existing strategies for personalized intervention are of interest. Routinely processed material from 246 untreated COADs with clinical follow-up was probed for evidence of DNA damage response (DDR), that is, the gathering of DDR-associated molecules at discrete nuclear spots, by multiplex immunofluorescence and immunohistochemical staining for DDR complex proteins (γH2AX, pCHK2, and pNBS1). We also tested the cases for type I interferon response, T-lymphocyte infiltration (TILs), and mutation mismatch repair defects (MMRd), known to be associated with defects of DNA repair. FISH analysis for chromosome 20q copy number variations was obtained. A total of 33.7% of COAD display a coordinated DDR on quiescent, non-senescent, non-apoptotic glands, irrespective of TP53 status, chromosome 20q abnormalities, and type I IFN response. Clinicopathological parameters did not differentiate DDR+ cases from the other cases. TILs were equally present in DDR and non-DDR cases. DDR+ MMRd cases were preferentially retaining wild-type MLH1. The outcome after 5FU-based chemotherapy was not different in the two groups. DDR+ COAD represents a subgroup not aligned with known diagnostic, prognostic, or therapeutic categories, with potential new targeted treatment opportunities, exploiting the DNA damage repair pathways.

BRAQUE: Bayesian Reduction for Amplified Quantization in UMAP Embedding.

Single-cell biology has revolutionized the way we understand biological processes. In this paper, we provide a more tailored approach to clustering and analyzing spatial single-cell data coming from immunofluorescence imaging techniques. We propose Bayesian Reduction for Amplified Quantization in UMAP Embedding (BRAQUE) as an integrative novel approach, from data preprocessing to phenotype classification. BRAQUE starts with an innovative preprocessing, named Lognormal Shrinkage, which is able to enhance input fragmentation by fitting a lognormal mixture model and shrink each component towards its median, in order to help further the clustering step in finding more separated and clear clusters. Then, BRAQUE's pipeline consists of a dimensionality reduction step performed using UMAP, and a clustering performed using HDBSCAN on UMAP embedding. In the end, clusters are assigned to a cell type by experts, using effects size measures to rank markers and identify characterizing markers (Tier 1), and possibly characterize markers (Tier 2). The number of total cell types in one lymph node detectable with these technologies is unknown and difficult to predict or estimate. Therefore, with BRAQUE, we achieved a higher granularity than other similar algorithms such as PhenoGraph, following the idea that merging similar clusters is easier than splitting unclear ones into clear subclusters.

Automated multimodal fluorescence microscopy for hyperplex spatial-proteomics: Coupling microfluidic-based immunofluorescence to high resolution, high sensitivity, three-dimensional analysis of histological slides.

In situ multiplexing analysis and in situ transcriptomics are now providing revolutionary tools to achieve the comprehension of the molecular basis of cancer and to progress towards personalized medicine to fight the disease. The complexity of these tasks requires a continuous interplay among different technologies during all the phases of the experimental procedures. New tools are thus needed and their characterization in terms of performances and limits is mandatory to reach the best resolution and sensitivity. We propose here a new experimental pipeline to obtain an optimized costs-to-benefits ratio thanks to the alternate employment of automated and manual procedures during all the phases of a multiplexing experiment from sample preparation to image collection and analysis. A comparison between ultra-fast and automated immunofluorescence staining and standard staining protocols has been carried out to compare the performances in terms of antigen saturation, background, signal-to-noise ratio and total duration. We then developed specific computational tools to collect data by automated analysis-driven fluorescence microscopy. Computer assisted selection of targeted areas with variable magnification and resolution allows employing confocal microscopy for a 3D high resolution analysis. Spatial resolution and sensitivity were thus maximized in a framework where the amount of stored data and the total requested time for the procedure were optimized and reduced with respect to a standard experimental approach.

Next-Generation Pathology Using Multiplexed Immunohistochemistry: Mapping Tissue Architecture at Single-Cell Level.

Single-cell omics aim at charting the different types and properties of all cells in the human body in health and disease. Over the past years, myriads of cellular phenotypes have been defined by methods that mostly required cells to be dissociated and removed from their original microenvironment, thus destroying valuable information about their location and interactions. Growing insights, however, are showing that such information is crucial to understand complex disease states. For decades, pathologists have interpreted cells in the context of their tissue using low-plex antibody- and morphology-based methods. Novel technologies for multiplexed immunohistochemistry are now rendering it possible to perform extended single-cell expression profiling using dozens of protein markers in the spatial context of a single tissue section. The combination of these novel technologies with extended data analysis tools allows us now to study cell-cell interactions, define cellular sociology, and describe detailed aberrations in tissue architecture, as such gaining much deeper insights in disease states. In this review, we provide a comprehensive overview of the available technologies for multiplexed immunohistochemistry, their advantages and challenges. We also provide the principles on how to interpret high-dimensional data in a spatial context. Similar to the fact that no one can just "read" a genome, pathological assessments are in dire need of extended digital data repositories to bring diagnostics and tissue interpretation to the next level.

Dissecting the histological features of lupus nephritis highlights new common patterns of injury in class III/IV.

OBJECTIVE: The International Society of Nephrology/Renal Pathology Society classification is the gold standard for the characterisation of lupus nephritis (LN) on renal biopsy, with therapeutic repercussions. Its recent revision simplified the current class subdivisions, eliminating the S/G forms of class IV, although data on a possible pathogenetic/clinical value of this subdivision are still contradictory. METHODS: 353 renal biopsies from Belimumab International Study in LN were assessed through central pathology review. Univariate logistic models and a decision tree were performed on 314 adequate biopsies to evaluate the impact of histological features on focal/diffuse classes. Removing class I/II (n=6) and 'pure' class V (n=34), principal component analysis (PCA) and heatmap were used to explore similarities among III, IVS and IVG biopsies either incorporating or not the mixed classes (+V, n=274). Finally, a method aimed at partitioning the cases into k clusters based on their similarity (KMeans), was used to study features from the cohort of 'pure' class III/IVS/IVG cases (n=214) to determine alternative subdivisions based on phenotypic data. RESULTS: Segmental endocapillary hypercellularity (EH) was prevalent in class III, global EH, wire loops, hyaline thrombi and double contours were hallmarks of class IVG, with IVS cases showing intermediate characteristics. Heatmap and PCA confirmed the segregation of these features among classes, showing better segregation for focal/diffuse LN as compared with the mixed classes (+V). KMeans revealed the presence of two main clusters, membranoproliferative-like (n=83) or vasculitis-like (n=131). CONCLUSIONS: This study reveals new phenotypic forms of LN surpassing the traditional classes as determined by the current classification. Future validation and confirmation are required to confirm these findings.

Mapping the Immune Landscape in Metastatic Melanoma Reveals Localized Cell-Cell Interactions That Predict Immunotherapy Response.

While immune checkpoint-based immunotherapy (ICI) shows promising clinical results in patients with cancer, only a subset of patients responds favorably. Response to ICI is dictated by complex networks of cellular interactions between malignant and nonmalignant cells. Although insights into the mechanisms that modulate the pivotal antitumoral activity of cytotoxic T cells (Tcy) have recently been gained, much of what has been learned is based on single-cell analyses of dissociated tumor samples, resulting in a lack of critical information about the spatial distribution of relevant cell types. Here, we used multiplexed IHC to spatially characterize the immune landscape of metastatic melanoma from responders and nonresponders to ICI. Such high-dimensional pathology maps showed that Tcy gradually evolve toward an exhausted phenotype as they approach and infiltrate the tumor. Moreover, a key cellular interaction network functionally linked Tcy and PD-L1+ macrophages. Mapping the respective spatial distributions of these two cell populations predicted response to anti-PD-1 immunotherapy with high confidence. These results suggest that baseline measurements of the spatial context should be integrated in the design of predictive biomarkers to identify patients likely to benefit from ICI. SIGNIFICANCE: This study shows that spatial characterization can address the challenge of finding efficient biomarkers, revealing that localization of macrophages and T cells in melanoma predicts patient response to ICI. See related commentary by Smalley and Smalley, p. 3198.

The normal and fibrotic mouse lung classified by spatial proteomic analysis.

Single cell classification is elucidating homeostasis and pathology in tissues and whole organs. We applied in situ spatial proteomics by multiplex antibody staining to routinely processed mouse lung, healthy and during a fibrosis model. With a limited validated antibody panel (24) we classify the normal constituents (alveolar type I and II, bronchial epithelia, endothelial, muscular, stromal and hematopoietic cells) and by quantitative measurements, we show the progress of lung fibrosis over a 4 weeks course, the changing landscape and the cell-specific quantitative variation of a multidrug transporter. An early decline in AT2 alveolar cells and a progressive increase in stromal cells seems at the core of the fibrotic process.

DNA Damage in Circulating Hematopoietic Progenitor Stem Cells as Promising Biological Sensor of Frailty.

Frailty is an age-related syndrome that exposes individuals to increased vulnerability. Although it is potentially reversible, in most cases it leads to negative outcomes, including mortality. The different methods proposed identify frailty after the onset of clinical manifestations. An early diagnosis might make it possible to manage the frailty progression better. The frailty pathophysiology is still unclear although mechanisms, in particular, those linked to inflammation and immunosenescence, have been investigated. A common feature of several clinical aspects involved in senescent organisms is the increase of oxidative stress, described as one of the major causes of deoxyribonucleic acid (DNA) damage accumulation in aged cells including the adult stem cell compartment. Likely, this accumulation is implicated in frailty status. The oxidative status of our frail, pre-frail, and non-frail population was characterized. In addition, the DNA damage in hematopoietic cells was evidenced by analyzing the peripheral blood mononuclear cell and their T lymphocyte, monocyte, circulating hematopoietic progenitor stem cell (cHPSC) subpopulations. The phosphorylation of C-terminal of histone H2AX at amino acid Ser 139 (γ-H2AX), which occurs at the DNA double-strand break focus, was evaluated. In our frail population, increased oxidative stress and a high level of DNA damage in cHPSC were found. This study may have potential implications because the increment of DNA damage in cHPSC could be suggestive of an organism impairment preceding the evident frailty. In addition, it may open the possibility for attenuation of frailty progression throughout specific drugs acting on preventing DNA damage or removing damaged cells.

Rejuvenated Vintage Tissue Sections Highlight Individual Antigen Fate During Processing and Long-term Storage.

Antigen-bearing proteins become progressively unavailable to immunodetection after prolonged storage of routine sections, exposed to a variety of agents, such as moisture, oxygen, and temperature. By proteomic analysis, the antigens are retained in the sections and definitely in the tissue block, pointing to fixation-independent, storage time-dependent protein modifications. Based on previous experience, we hypothesized that a combined exposure to a reducing agent and to chemicals favoring protein conformation changes would reverse the masking in aged sections. Disaccharides, lactose and sucrose, and a surfactant, added to a standard antigen retrieval buffer, reverse the negative changes in aged sections. Furthermore, they provide enhanced access to antigens in freshly cut sections, but not universally, revealing additional factors, besides heat and calcium chelation, required for antigen retrieval of individual proteins.

Lipidomic Typing of Colorectal Cancer Tissue Containing Tumour-Infiltrating Lymphocytes by MALDI Mass Spectrometry Imaging.

Predicting the prognosis of colorectal cancer (CRC) patients remains challenging and a characterisation of the tumour immune environment represents one of the most crucial avenues when attempting to do so. For this reason, molecular approaches which are capable of classifying the immune environments associated with tumour infiltrating lymphocytes (TILs) are being readily investigated. In this proof of concept study, we aim to explore the feasibility of using spatial lipidomics by MALDI-MSI to distinguish CRC tissue based upon their TIL content. Formalin-fixed paraffin-embedded tissue from human thymus and tonsil was first analysed by MALDI-MSI to obtain a curated mass list from a pool of single positive T lymphocytes, whose putative identities were annotated using an LC-MS-based lipidomic approach. A CRC tissue microarray (TMA, n = 30) was then investigated to determine whether these cases could be distinguished based upon their TIL content in the tumour and its microenvironment. MALDI-MSI from the pool of mature T lymphocytes resulted in the generation of a curated mass list containing 18 annotated m/z features. Initially, subsets of T lymphocytes were then distinguished based on their state of maturation and differentiation in the human thymus and tonsil tissue. Then, when applied to a CRC TMA containing differing amounts of T lymphocyte infiltration, those cases with a high TIL content were distinguishable from those with a lower TIL content, especially within the tumour microenvironment, with three lipid signals being shown to have the greatest impact on this separation (p < 0.05). On the whole, this preliminary study represents a promising starting point and suggests that a lipidomics MALDI-MSI approach could be a promising tool for subtyping the diverse immune environments in CRC.

Background-free Detection of Mouse Antibodies on Mouse Tissue by Anti-isotype Secondary Antibodies.

Immunodetection on mouse routinely processed tissue via antibodies raised in mice faces cross-reactivity of the secondary anti-mouse reagents with endogenous immunoglobulins, which permeate the tissue. Various solutions to this problem have been devised and include endogenous Ig block with anti-mouse Fab fragments or directly conjugated primary antibodies. Mouse isotype-specific antibodies, differently from reagents directed against both heavy and light chains, fail to detect endogenous Ig after fixation and embedding, while providing a clean and specific detection system for mouse antibodies on mouse routinely processed tissue.

A Multi-Omics Analysis of Metastatic Melanoma Identifies a Germinal Center-Like Tumor Microenvironment in HLA-DR-Positive Tumor Areas.

The emergence of immune checkpoint inhibitors has dramatically changed the therapeutic landscape for patients with advanced melanoma. However, relatively low response rates and a high incidence of severe immune-related adverse events have prompted the search for predictive biomarkers. A positive predictive value has been attributed to the aberrant expression of Human Leukocyte Antigen-DR (HLA-DR) by melanoma cells, but it remains unknown why this is the case. In this study, we have examined the microenvironment of HLA-DR positive metastatic melanoma samples using a multi-omics approach. First, using spatial, single-cell mapping by multiplexed immunohistochemistry, we found that the microenvironment of HLA-DR positive melanoma regions was enriched by professional antigen presenting cells, including classical dendritic cells and macrophages, while a more general cytotoxic T cell exhaustion phenotype was present in these regions. In parallel, transcriptomic analysis on micro dissected tissue from HLA-DR positive and HLA-DR negative areas showed increased IFNγ signaling, enhanced leukocyte adhesion and mononuclear cell proliferation in HLA-DR positive areas. Finally, multiplexed cytokine profiling identified an increased expression of germinal center cytokines CXCL12, CXCL13 and CCL19 in HLA-DR positive metastatic lesions, which, together with IFNγ and IL4 could serve as biomarkers to discriminate tumor samples containing HLA-DR overexpressing tumor cells from HLA-DR negative samples. Overall, this suggests that HLA-DR positive areas in melanoma attract the anti-tumor immune cell infiltration by creating a dystrophic germinal center-like microenvironment where an enhanced antigen presentation leads to an exhausted microenvironment, nevertheless representing a fertile ground for a better efficacy of anti-PD-1 inhibitors due to simultaneous higher levels of PD-1 in the immune cells and PD-L1 in the HLA-DR positive melanoma cells.

Antibodies validated for routinely processed tissues stain frozen sections unpredictably.

Background: Antibody validation for tissue staining is required for reproducibility; criteria to ensure validity have been published recently. The majority of these recommendations imply the use of routinely processed (formalin-fixed, paraffin-embedded) tissue. Materials & methods: We applied to lightly fixed frozen sections a panel of 126 antibodies validated for formalin-fixed, paraffin-embedded tissue with extended criteria. Results: Less than 30% of the antibodies performed as expected with all fixations. 35% preferred one fixation over another, 13% gave nonspecific staining and 23% did not stain at all. Conclusion: Individual antibody variability of the paratope's fitness for the fixed antigen may be the cause. Revalidation of established antibody panels is required when they are applied to sections whose fixation and processing are different from the tissue where they were initially validated.

Proteomics for the study of new biomarkers in Fabry disease: State of the art.

Nephropathy represents a major complication of Fabry Disease and its accurate characterization is of paramount importance in predicting the disease progression and assessing the therapeutic responses. The diagnostic process still relies on performing renal biopsy, nevertheless many efforts have been made to discover early reliable biomarkers allowing us to avoid invasive procedures. In this field, proteomics offers a sensitive and fast method leading to an accurate detection of specific pathological proteins and the discovery of diagnostic and prognostic biomarkers that reflect disease progression and facilitate the evaluation of therapeutic responses. Here, we report a review of selected literature focusing on the investigation of several proteomic techniques highlighting their advantages, limitations and future perspectives in their application in the routine study of Fabry Nephropathy.

PKHhigh/CD133+/CD24- Renal Stem-Like Cells Isolated from Human Nephrospheres Exhibit In Vitro Multipotency.

The mechanism upon which human kidneys undergo regeneration is debated, though different lineage-tracing mouse models have tried to explain the cellular types and the mechanisms involved. Different sources of human renal progenitors have been proposed, but it is difficult to argue whether these populations have the same capacities that have been described in mice. Using the nephrosphere (NS) model, we isolated the quiescent population of adult human renal stem-like PKHhigh/CD133+/CD24- cells (RSC). The aim of this study was to deepen the RSC in vitro multipotency capacity. RSC, not expressing endothelial markers, generated secondary nephrospheres containing CD31+/vWf+ cells and cytokeratin positive cells, indicating the coexistence of endothelial and epithelial commitment. RSC cultured on decellularized human renal scaffolds generated endothelial structures together with the proximal and distal tubular structures. CD31+ endothelial committed progenitors sorted from nephrospheres generated spheroids with endothelial-like sprouts in Matrigel. We also demonstrated the double commitment toward endothelial and epithelial lineages of single RSC. The ability of the plastic RSC population to recapitulate the development of tubular epithelial and endothelial renal lineages makes these cells a good tool for the creation of organoids with translational relevance for studying the parenchymal and endothelial cell interactions and developing new therapeutic strategies.