Impact of Decalcification, Cold Ischemia, and Deglycosylation on Performance of Programmed Cell Death Ligand-1 Antibodies With Different Binding Epitopes: Comparison of 7 Clones.

Programmed cell death ligand-1 (PD-L1) expression levels in patients' tumors have demonstrated clinical utility across many cancer types and are used to determine treatment eligibility. Several independently developed PD-L1 immunohistochemical (IHC) predictive assays are commercially available and have demonstrated different levels of staining between assays, generating interest in understanding the similarities and differences between assays. Previously, we identified epitopes in the internal and external domains of PD-L1, bound by antibodies in routine clinical use (SP263, SP142, 22C3, and 28-8). Variance in performance of assays utilizing these antibodies, observed following exposure to preanalytical factors such as decalcification, cold ischemia, and duration of fixation, encouraged additional investigation of antibody-binding sites, to understand whether binding site structures/conformations contribute to differential PD-L1 IHC assay staining. We proceeded to further investigate the epitopes on PD-L1 bound by these antibodies, alongside the major clones utilized in laboratory-developed tests (E1L3N, QR1, and 73-10). Characterization of QR1 and 73-10 clones demonstrated that both bind the PD-L1 C-terminal internal domain, similar to SP263/SP142. Our results also demonstrate that under suboptimal decalcification or fixation conditions, the performance of internal domain antibodies is less detrimentally affected than that of external domain antibodies 22C3/28-8. Furthermore, we show that the binding sites of external domain antibodies are susceptible to deglycosylation and conformational structural changes, which directly result in IHC staining reduction or loss. The binding sites of internal domain antibodies were unaffected by deglycosylation or conformational structural change. This study demonstrates that the location and conformation of binding sites, recognized by antibodies employed in PD-L1 diagnostic assays, differ significantly and exhibit differing degrees of robustness. These findings should reinforce the need for vigilance when performing clinical testing with different PD-L1 IHC assays, particularly in the control of cold ischemia and the selection of fixation and decalcification conditions.

Usability of deep learning and H&E images predict disease outcome-emerging tool to optimize clinical trials.

Understanding factors that impact prognosis for cancer patients have high clinical relevance for treatment decisions and monitoring of the disease outcome. Advances in artificial intelligence (AI) and digital pathology offer an exciting opportunity to capitalize on the use of whole slide images (WSIs) of hematoxylin and eosin (H&E) stained tumor tissue for objective prognosis and prediction of response to targeted therapies. AI models often require hand-delineated annotations for effective training which may not be readily available for larger data sets. In this study, we investigated whether AI models can be trained without region-level annotations and solely on patient-level survival data. We present a weakly supervised survival convolutional neural network (WSS-CNN) approach equipped with a visual attention mechanism for predicting overall survival. The inclusion of visual attention provides insights into regions of the tumor microenvironment with the pathological interpretation which may improve our understanding of the disease pathomechanism. We performed this analysis on two independent, multi-center patient data sets of lung (which is publicly available data) and bladder urothelial carcinoma. We perform univariable and multivariable analysis and show that WSS-CNN features are prognostic of overall survival in both tumor indications. The presented results highlight the significance of computational pathology algorithms for predicting prognosis using H&E stained images alone and underpin the use of computational methods to improve the efficiency of clinical trial studies.

Impairment of Glycolysis-Derived l-Serine Production in Astrocytes Contributes to Cognitive Deficits in Alzheimer's Disease.

Alteration of brain aerobic glycolysis is often observed early in the course of Alzheimer's disease (AD). Whether and how such metabolic dysregulation contributes to both synaptic plasticity and behavioral deficits in AD is not known. Here, we show that the astrocytic l-serine biosynthesis pathway, which branches from glycolysis, is impaired in young AD mice and in AD patients. l-serine is the precursor of d-serine, a co-agonist of synaptic NMDA receptors (NMDARs) required for synaptic plasticity. Accordingly, AD mice display a lower occupancy of the NMDAR co-agonist site as well as synaptic and behavioral deficits. Similar deficits are observed following inactivation of the l-serine synthetic pathway in hippocampal astrocytes, supporting the key role of astrocytic l-serine. Supplementation with l-serine in the diet prevents both synaptic and behavioral deficits in AD mice. Our findings reveal that astrocytic glycolysis controls cognitive functions and suggest oral l-serine as a ready-to-use therapy for AD.

Fast fluorescence in situ hybridisation for the enhanced detection of MET in non-small cell lung cancer.

The c-Met/hepatocyte growth factor receptor pathway is frequently dysregulated in multiple cancer types, including non-small cell lung cancer (NSCLC). MET amplification has been shown to develop as a resistance mechanism to treatment in NSCLC. The identification of increased MET copy number within tumour cells is increasingly important to stratify those tumours and patients which are susceptible to treatment targetting MET kinase inhibition. Fluorescence in situ hybridisation (FISH) has been successfully employed to identify patients with abnormal MET gene copy number with numerous probes available for use. Here we report a FISH protocol that reduces probe hybridisation time in NSCLC tissue to 1 hour and compare the results with other protocols. MET gene copy number was determined in 20 NSCLC cases using 3 FISH probes: 1. Kreatech FISH, MET (7q31) SE 7 ready to use probes, hybridised using an overnight protocol; 2. Dako MET IQFISH probe with CEP7 ready to use probe, hybridised for 2 hours; 3. Kreatech MET (7q31) SE 7 XL FISH probe, prepared in SwiftFISH buffer and hybridised for 1 hour. The MET gene copy number and MET: centromere 7 gene ratio were determined for each tissue and cases categorised as having MET high or MET low status. All three FISH probes were shown to demonstrate good agreement with each other. Overall percentage agreement between probes was ≥90%. Intraclass correlation showed good agreement (ICC ≥0.80) between all three assays for MET gene copy number and MET: centromere 7 gene ratio. These FISH protocols provide evidence that rapid laboratory developed FISH assays with short turnaround time perform consistently with standard protocols, potentially enabling faster treatment decisions.

Voxel-Based Statistical Analysis of 3D Immunostained Tissue Imaging.

Recently developed techniques to visualize immunostained tissues in 3D and in large samples have expanded the scope of microscopic investigations at the level of the whole brain. Here, we propose to adapt voxel-based statistical analysis to 3D high-resolution images of the immunostained rodent brain. The proposed approach was first validated with a simulation dataset with known cluster locations. Then, it was applied to characterize the effect of ADAM30, a gene involved in the metabolism of the amyloid precursor protein, in a mouse model of Alzheimer's disease. This work introduces voxel-based analysis of 3D immunostained microscopic brain images and, therefore, opens the door to localized whole-brain exploratory investigation of pathological markers and cellular alterations.

Tumour Heterogeneity of Breast Cancer: From Morphology to Personalised Medicine.

Breast cancer (BC) displays striking clinical, morphological, and behavioural diversity within a single tumour and between tumours. Currently, mounting evidence indicates that the morphological heterogeneity of BC reflects an underlying spectrum of genetic and epigenetic portraits that control BC behaviour. Further understanding of BC heterogeneity will have an impact, not only on the routine diagnostic practices but also on patients' management decisions. Phenomena like diagnostic inconsistencies and therapeutic resistance, both primary and acquired, could be attributed, at least in part, to tumour heterogeneity within the same cancer and between the primary disease and subsequent recurrences. From a practical standpoint, and to minimise the impact of BC intratumoral heterogeneity, pragmatic approaches for adequate tumour sampling have been suggested in translational biomarker discovery and validation research studies and in the clinical setting. Here, we provide a brief overview of BC heterogeneity, with an emphasis on the clinical consequences of intratumoral heterogeneity.

A validation dataset for Macaque brain MRI segmentation.

Validation data for segmentation algorithms dedicated to preclinical images is fiercely lacking, especially when compared to the large number of databases of Human brain images and segmentations available to the academic community. Not only is such data essential for validating methods, it is also needed for objectively comparing concurrent algorithms and detect promising paths, as segmentation challenges have shown for clinical images. The dataset we present here is a first step in this direction. It comprises 10 T2-weighted MRIs of healthy adult macaque brains, acquired on a 7 T magnet, along with corresponding manual segmentations into 17 brain anatomic labelled regions spread over 5 hierarchical levels based on a previously published macaque atlas (Calabrese et al., 2015) [1]. By giving access to this unique dataset, we hope to provide a reference needed by the non-human primate imaging community. This dataset was used in an article presenting a new primate brain morphology analysis pipeline, Primatologist (Balbastre et al., 2017) [2]. Data is available through a NITRC repository (https://www.nitrc.org/projects/mircen_macset).

Primatologist: A modular segmentation pipeline for macaque brain morphometry.

Because they bridge the genetic gap between rodents and humans, non-human primates (NHPs) play a major role in therapy development and evaluation for neurological disorders. However, translational research success from NHPs to patients requires an accurate phenotyping of the models. In patients, magnetic resonance imaging (MRI) combined with automated segmentation methods has offered the unique opportunity to assess in vivo brain morphological changes. Meanwhile, specific challenges caused by brain size and high field contrasts make existing algorithms hard to use routinely in NHPs. To tackle this issue, we propose a complete pipeline, Primatologist, for multi-region segmentation. Tissue segmentation is based on a modular statistical model that includes random field regularization, bias correction and denoising and is optimized by expectation-maximization. To deal with the broad variety of structures with different relaxing times at 7 T, images are segmented into 17 anatomical classes, including subcortical regions. Pre-processing steps insure a good initialization of the parameters and thus the robustness of the pipeline. It is validated on 10 T2-weighted MRIs of healthy macaque brains. Classification scores are compared with those of a non-linear atlas registration, and the impact of each module on classification scores is thoroughly evaluated.

Relevance of deep learning to facilitate the diagnosis of HER2 status in breast cancer.

Tissue biomarker scoring by pathologists is central to defining the appropriate therapy for patients with cancer. Yet, inter-pathologist variability in the interpretation of ambiguous cases can affect diagnostic accuracy. Modern artificial intelligence methods such as deep learning have the potential to supplement pathologist expertise to ensure constant diagnostic accuracy. We developed a computational approach based on deep learning that automatically scores HER2, a biomarker that defines patient eligibility for anti-HER2 targeted therapies in breast cancer. In a cohort of 71 breast tumour resection samples, automated scoring showed a concordance of 83% with a pathologist. The twelve discordant cases were then independently reviewed, leading to a modification of diagnosis from initial pathologist assessment for eight cases. Diagnostic discordance was found to be largely caused by perceptual differences in assessing HER2 expression due to high HER2 staining heterogeneity. This study provides evidence that deep learning aided diagnosis can facilitate clinical decision making in breast cancer by identifying cases at high risk of misdiagnosis.

ADAM30 Downregulates APP-Linked Defects Through Cathepsin D Activation in Alzheimer's Disease.

Although several ADAMs (A disintegrin-like and metalloproteases) have been shown to contribute to the amyloid precursor protein (APP) metabolism, the full spectrum of metalloproteases involved in this metabolism remains to be established. Transcriptomic analyses centred on metalloprotease genes unraveled a 50% decrease in ADAM30 expression that inversely correlates with amyloid load in Alzheimer's disease brains. Accordingly, in vitro down- or up-regulation of ADAM30 expression triggered an increase/decrease in Aß peptides levels whereas expression of a biologically inactive ADAM30 (ADAM30(mut)) did not affect Aß secretion. Proteomics/cell-based experiments showed that ADAM30-dependent regulation of APP metabolism required both cathepsin D (CTSD) activation and APP sorting to lysosomes. Accordingly, in Alzheimer-like transgenic mice, neuronal ADAM30 over-expression lowered Aß42 secretion in neuron primary cultures, soluble Aß42 and amyloid plaque load levels in the brain and concomitantly enhanced CTSD activity and finally rescued long term potentiation alterations. Our data thus indicate that lowering ADAM30 expression may favor Aß production, thereby contributing to Alzheimer's disease development.

In Vivo Detection of Amyloid Plaques by Gadolinium-Stained MRI Can Be Used to Demonstrate the Efficacy of an Anti-amyloid Immunotherapy.

Extracellular deposition of ß amyloid plaques is an early event associated to Alzheimer's disease. Here, we have used in vivo gadolinium-stained high resolution (29(∗)29(∗)117 µm(3)) magnetic resonance imaging (MRI) to follow-up in a longitudinal way individual amyloid plaques in APP/PS1 mice and evaluate the efficacy of a new immunotherapy (SAR255952) directed against protofibrillar and fibrillary forms of Aß. APP/PS1 mice were treated for 5 months between the age of 3.5 and 8.5 months. SAR255952 reduced amyloid load in 8.5-months-old animals, but not in 5.5-months animals compared to mice treated with a control antibody (DM4). Histological evaluation confirmed the reduction of amyloid load and revealed a lower density of amyloid plaques in 8.5-months SAR255952-treated animals. The longitudinal follow-up of individual amyloid plaques by MRI revealed that plaques that were visible at 5.5 months were still visible at 8.5 months in both SAR255952 and DM4-treated mice. This suggests that the amyloid load reduction induced by SAR255952 is related to a slowing down in the formation of new plaques rather than to the clearance of already formed plaques.

High-throughput 3D whole-brain quantitative histopathology in rodents.

Histology is the gold standard to unveil microscopic brain structures and pathological alterations in humans and animal models of disease. However, due to tedious manual interventions, quantification of histopathological markers is classically performed on a few tissue sections, thus restricting measurements to limited portions of the brain. Recently developed 3D microscopic imaging techniques have allowed in-depth study of neuroanatomy. However, quantitative methods are still lacking for whole-brain analysis of cellular and pathological markers. Here, we propose a ready-to-use, automated, and scalable method to thoroughly quantify histopathological markers in 3D in rodent whole brains. It relies on block-face photography, serial histology and 3D-HAPi (Three Dimensional Histology Analysis Pipeline), an open source image analysis software. We illustrate our method in studies involving mouse models of Alzheimer's disease and show that it can be broadly applied to characterize animal models of brain diseases, to evaluate therapeutic interventions, to anatomically correlate cellular and pathological markers throughout the entire brain and to validate in vivo imaging techniques.

Robust supervised segmentation of neuropathology whole-slide microscopy images.

Alzheimer's disease is characterized by brain pathological aggregates such as Aß plaques and neurofibrillary tangles which trigger neuroinflammation and participate to neuronal loss. Quantification of these pathological markers on histological sections is widely performed to study the disease and to evaluate new therapies. However, segmentation of neuropathology images presents difficulties inherent to histology (presence of debris, tissue folding, non-specific staining) as well as specific challenges (sparse staining, irregular shape of the lesions). Here, we present a supervised classification approach for the robust pixel-level classification of large neuropathology whole slide images. We propose a weighted form of Random Forest in order to fit nonlinear decision boundaries that take into account class imbalance. Both color and texture descriptors were used as predictors and model selection was performed via a leave-one-image-out cross-validation scheme. Our method showed superior results compared to the current state of the art method when applied to the segmentation of Aß plaques and neurofibrillary tangles in a human brain sample. Furthermore, using parallel computing, our approach easily scales-up to large gigabyte-sized images. To show this, we segmented a whole brain histology dataset of a mouse model of Alzheimer's disease. This demonstrates our method relevance as a routine tool for whole slide microscopy images analysis in clinical and preclinical research settings.