Machine learning in computational histopathology: Challenges and opportunities

Digital histopathological images, high‐resolution images of stained tissue samples, are a vital tool for clinicians to diagnose and stage cancers. The visual analysis of patient state based on these images are an important part of oncology workflow. Although pathology workflows have historically been conducted in laboratories under a microscope, the increasing digitization of histopathological images has led to their analysis on computers in the clinic. The last decade has seen the emergence of machine learning, and deep learning in particular, a powerful set of tools for the analysis of histopathological images. Machine learning models trained on large datasets of digitized histopathology slides have resulted in automated models for prediction and stratification of patient risk. In this review, we provide context for the rise of such models in computational histopathology, highlight the clinical tasks they have found success in automating, discuss the various machine learning techniques that have been applied to this domain, and underscore open problems and opportunities.


| INTRODUCTION
Anatomic pathology has undergone many important evolutions over the past centuries, from manual examination with bright-field microscopes to whole-slide imaging (WSI), computer vision and image analysis techniques, high-throughput molecular sequencing technologies and now artificial intelligence (AI). Since the development of the first microscope by Hans and Zacharias Janssen in 1590, 1 the microscope has been an important driving force for many discoveries in pathology, with the first few microscopic analysis of human tissue by Marcello Malpighi, 2 Anton van Leeuwenhoek, 3 and Johannes Muller 4 in the 16th and 17th centuries, theory of cell biology and cancer origin by Rudolf Virchow in 1855, 5,6 and the concept of recording histopathological characteristics (e.g., features) to make diagnoses such as the Reed-Sternberg cell for Hodgkin lymphoma in 1898/1902. [7][8][9][10] Computerbased analyses did not emerge until 1966 when Prewitt and Mendelson used image analysis algorithms to extract quantitative features for cell subtyping in blood smears. 11 The 1990s and following decade also marked a pivotal period in which commercial slide scanners were developed that could digitize histology slides into high-resolution WSIs, [12][13][14][15][16] as well as computer-aided diagnosis (CAD) systems that implemented early machine learning (ML) techniques using handcrafted cell and tissue features from WSIs. [17][18][19][20][21][22][23][24] In 2010, Dundar et al. presented the first formulation of multiple instance learning (MIL) for cancer subtyping in WSIs based on Haralick texture features, a framework that is still widely utilized today in performing weakly-supervised cancer diagnosis at scale without needing detailed pathologist annotations. 25 Over the past half decade and still ongoing, the emergence of AI and ML, via deep learning, has been the latest driving force for advancements in pathology. 26 Following the 2012 success of Michael Cooper, Zongliang Ji, and Rahul G. Krishnan contributed equally to this study. convolutional neural networks (CNNs) in the ImageNet Large Scale Visual Recognition Challenge, [27][28][29] similar open challenges such as the CAMELYON 30,31 and PANDA 32 challenges were created for lymph node metastasis detection and Gleason grading in prostate cancer, respectively, which led to important breakthroughs showing that CNNand multiple-instance learning (MIL)-based classification systems could surpass pathologist-level performance on these diagnostic tasks. 33 Given a large enough repository of diagnostic WSIs (n > 100), 34 deep learning can be used to formulate and solve new clinical tasks beyond human pathologist capabilities such as metastatic origin of cancer prediction, 35 cancer prognostication, [36][37][38][39][40] and microsatellite instability (MSI) prediction. [41][42][43] Looking beyond supervised learning applications via CNNs and MIL, the development of other techniques such as generative AI modeling, [44][45][46] geometric deep learning, 47,48 unsupervised learning, 49,50 and multimodal deep learning 51 may soon enable new clinical capabilities that could enter pathology and laboratory medicine workflows, such as virtual staining, [52][53][54] elucidating cell and tissue interactions, 55 untargeted biomarker discovery, 56,57 data fusion with genomics and other rich biomedical data streams. [58][59][60][61] Though direct application of many deep learning techniques may appear to work "out-of-the-box" and have emergent capabilities in computational pathology (CPATH), there exists a variety of technical challenges that would limit their adoption in clinical translation, deployment, and commercialization. Compared to natural images, WSI as a computer vision domain can be much more challenging due to the multi-pyramidal, gigapixel image resolutions of the WSI digital format, which can add cost in annotating regions-of-interests, finding data storage, and running image analysis pipelines. For slide-level cancer diagnosis tasks, established approaches can overcome these limitations, such as employing a pretrained (CNN) to pre-extract features from nonoverlapping (e.g., 256 Â 256 image resolution at 20Â, 40Â) tissue patches in the WSI, and then inputting the pre-extracted features into a downstream MIL framework. 34 However, depending on the task, choices regarding patch image resolution, patch image magnification and spatial ordering of patch features would strongly influence model performance. Many slide-level tasks in computational pathology also suffer from small support sizes, especially in rare diseases, which constrains approaches to also be lightweight and data-efficient. The effect of how genomics, ancestry, selfreported race, and other environmental factors can manifest as biases within pathology data is understudied and may have more important implications when such models are deployed across diverse populations. 62 Lastly, as many of these advances in computational pathology stem originally from advances made elsewhere in ML, computer vision and deep learning, limitations of these previous methods may still exist in their current application in pathology.
Computational pathology has received significant coverage from previous reviews and perspectives. References 75 fairness, 62 and individual cancer types. 75 In this review, we organize a technical overview of current deep learning applications to pathology, disentangling the clinical tasks from the key methodological tools in ML used to solve them. We highlight several open opportunities for CPATH in the context of surrounding discussions in fairness, equity, interpretability, and the rise of large language models (LLMs). Figure 1 presents a visual overview of this review enumerating the models surveyed spanning cancer types, different ML models, learning strategies and their trends across time.

| CLINICAL TASKS IN COMPUTATIONAL HISTOPATHOLOGY
Digitized WSIs are routinely read by pathologists to extract and acquire insights for diagnosing the current and future state of patient F I G U R E 1 A Sankey diagram of the experiments presented in the papers reviewed in this article. Each unit of height in the diagram represents a single experiment, comprising an anatomical region of the body, pathological task, machine learning methodology, and year of publication. This diagram allows visualization of the frequency of interactions between these characteristics of each experiment. Specifically, this figure highlights the increase in popularity of self-supervision, attention, and graph-based architectures in recent years, and the continued popularity of convolutional architectures and multiple-instance learning algorithms in this space.

| Classification
The winning solution 29 of ImageNet 2012 28 showed the capability of deep neural network to accurately classify natural images with large, labeled dataset. Given histopathological WSIs, the most common task that an ML model can help with is to tell whether the scanned tissue possesses abnormalities of interest. According to cancer data released by the World Health Organization, the five most common cancer types are breast cancer, lung cancer, colon cancer, prostate cancer, and stomach cancer. 87 Prior work in classification uses the word "detection" for certain diseases or disease subtypes. The task of detection in CPATH is different from the task of object detection in computer vision where images are significantly smaller and often contain a small number of objects. In CPATH, due to limited accessibility to bounding box or positional-level data, only a few studies in computational pathology 88,89,83,90,91 can do fine-grained detection of mitotic events. Consequently, the vast majority of research methods focus on predicting the prevalence of clinically relevant patient or slide level outcomes posed as a classification task. The intended goal of such predictive systems can range from automation of prediction in lowresource settings to risk stratification for organizing clinical workflows.
For breast cancer, a key task is understanding whether the WSI contains mitotic or non-mitotic cells, 89,92-102 or tumorous cells. 103,102 Studies have explored the use of ML to distinguish breast cancer WSIs between normal, benign, carcinoma in situ (CIS) and breast invasive carcinoma (BIC). 104 Classification has also been used to identify specific subtypes of immunostaining for estrogen, progesterone, and Her2 receptors (ER/PR/Her2), 105,106 Ductal or Lobular, Basal-like or non-Basal-like, and different tumor grades. 105 Two-stage classification, first predicting if a given WSI tile contains a tumor, and then identifying whether the tumor image patch contains tumor-infiltrating lymphocytes (TIL) has also been explored. 103 For lung cancer, an important goal is cancer subtyping into adenocarcinoma (LUAD) or squamous cell carcinoma (LUSC) 38,107 and distinguishing genetic mutations within subtypes. 38 Research has studied the identification of histologic subtypes like lepidic, acinar, papillary, micropapillary, and solid [108][109][110] and categorizing WSIs into PDL1-positive, and PDL1-negative. 111 For colon cancer, in addition to cancer or noncancer WSI classification, 36,43,[112][113][114][115]  CPATH has made inroads into predictive problems for diseases in the brain, liver and skin. For brain gilomas, researchers have used ML for grading WSIs and identifying if the tissue morphology indicates a IDH1 mutation. [128][129][130] For liver cancers, 131 research has studied classification in the context of 2-class ballooning, 3-class inflammation, 4-class steatosis, and 5-class fibrosis, 132 discriminating between two subtypes of primary liver cancer, hepatocellular carcinoma and cholangiocarcinoma, and built tools to assist real clinical workflows. 133 For bladder cancer, studies have focused on grading cancers using WSIs. 134,135 For skin cancer, a key task is identifying whether a given melanoma will recur based on a WSI. 136,137 Mesothelioma tissue WSIs were used to build classifier to identify transitional mesothelioma (TM) or not-TM tissue. 138,139 Finally, research has studied tumor versus non-tumor and TIL classification across different cancer types. 33

| Segmentation
While classification of clinical outcomes can provide utility at a slide level, ML has also been used to highlight the exact location and boundary of an abnormality in the WSI. This problem is typically posed as one of image segmentation. Pixel-level labels are required for models to perform segmentation tasks on histopathological images. However, pixel-level labels are hard to obtain since they would require pathologists to use software to draw boundaries around different type of tissues. Despite the effort required, researchers have made great progress in developing methods to automatically identify object of interests in histopathological images.
Due to the size of the WSI and the computational requirements of deep learning models, most methods split WSIs into patches where each patch has labeled segmentation masks (a binary mask over a pixel indicating which pixels represent regions of interest) for a model to learn. For colon cancer, research has studied the problem of segmenting glands 90,115,[144][145][146] and identifying different classes of tissues. 147 For kidney cancers, segmenting glomeruli 126,148 or different subtypes of kidney tissues 127,149 are key tasks of interest. Researchers have also made progress on segmenting normal and abnormal parts from histopathological images for breast, 90,99,150 lung, 110,151 bladder, 134 stomach, 123 prostate 90 cancer.

| Survival analysis
The successful prediction of patient outcomes such as mortality, and characteristics of their disease trajectory such as progression free survival, can help oncologists plan for treatments and assess individual patient disease severity. Histopathological images capture proxies of genetic abnormalities, tumor burden and subtype, all of which can inform this task. There are several studies that model patient trajectories using pathology data 36,39,[118][119][120][121]124,137 and those that blend clinical data with other data modalities such as demographic or genomics data. 40,[152][153][154] In most biomedical studies, the time-to-event for many patients is not recorded due to loss of patients to follow-up. Consequently, data often contains the last-observed time points for patients rather than their actual event time. Such data is referred to as (right) censored and the tools used to predict event time, typically time of death, given such data fall under the umbrella of survival analysis.
Since the combination of neural networks with classical tools in survival models, 155

| Counting
Clinicians are also interested in having detailed measurements of the sampled tissue at the cellular level. Counting mitotic cells is a typical clinical task for pathologists since the number of mitotic cells is a key factor for determining cancer grade. Research efforts have been made in counting mitosis cells in breast cancer, lymphocytes in breast cancer WSIs, centroblasts on follicular lymphoma WSIs, and plasma cells in bone marrow image patches. 41,64,83,164 Counting cells typically requires cell segmentation; a variety of attempts have been made for cell segmentation on breast cancer, [165][166][167][168] colon cancer, 146,169 and bladder cancer. 170 Research attempts have also been made on counting neuroendocrine tumor (NET) cells within the gastrointestinal tract and pancreas. 171 Cell nuclei segmentation and counting has also been developed for grading squamous epithelium cervical intraepithelial neoplasia (CIN). 172 Quantification of immunohistochemical labelling (e.g., MIB1/Ki-67 proliferation) is also an important prognostic application. [173][174][175] Although supervised cell segmentation requires tremendous effort to obtain fine-grained annotations, several cell segmentation methods 79,176,177 have obtained promising results. There have also been several unsupervised cell segmentation methods have been developed over the past decade. [178][179][180] There has also been research studying the use of ML for tasks that can form the basis for future clinical workflows. Many WSIs from tissue samples come without labels and obtaining such labels may be hard or impossible. Consequently, unsupervised ML methods like clustering have been deployed to obtain insights from histopathological images. Given image patches from WSIs, researchers first extract features, or representations, using preexisting predictive models. These are then clustered to automate the identification of subgroups in lung, 159 brain, 159 breast, 98,181 colon cancer 113 WSIs. In addition, research has begun to use ML to learn associations between gene expression and pathological images, 110,130,182 perform stain normalization for histopathological images, 106,183-186 generate synthetic data, 106,130,148,151,170 compress images 187 and automate histopathological captioning and diagnosis generation. 135

| LEARNING STRATEGIES FOR COMPUTATIONAL HISTOPATHOLOGY
The goal of an ML model developed to tackle the tasks in Section 2 is to generalize well, that is, it must operate in regimes outside of those in which it was trained. Deep learning models learn functions that transform high-dimensional inputs like images to numbers represent-

| Multiple-instance learning
Deep learning models require that the input (in this case, images) be small enough to load onto GPU memory. The need for MIL arises because, as of 2023, a single histopathology image would exceed the available memory of a GPU. A popular approach is to decompose a large histopathology image into patches and treat the bag of patches as a collection, all of which are assigned the same label. metastases. In evaluating generalization performance to external data, the authors conclude that weak supervision (e.g., slide-level labels) on larger datasets leads to better generalization than strong supervision (e.g., pixel-wise labels) on small datasets. Transfer learning refers to the class of methods that leverage parameters from a model trained on a prior task (also called an "upstream task") as a starting point for learning an eventual task of interest (also called the "downstream task"). In computer vision, certain parameters within a model often transfer well between imaging modalities and tasks. The weights of the first few layers of a deep CNN, for example, often learn edge detection capabilities, 195 which represents a common skill that is useful across many domains of computer vision task. It is therefore typical that a computer vision model that has been pretrained on an upstream image classification or regression task to be able to learn a novel data distribution more efficiently from fewer samples than a model that was trained from scratch. ImageNet, 27,28 a large-scale dataset of natural images, is a popular dataset for pretraining image recognition models, and ImageNetpretrained models are readily available as starting points for downstream image tasks. Despite clear visual differences between ImageNet's natural images and the fine-grained images of tissue morphology found in histopathology, pretrained models like VGG, 196 ResNet, 77 AlexNet, 29 GoogLeNet/Inception, 197,198 remain effective starting points for building predictive models in computational histopathology (VGG, 36,84,93 ResNet, 125 AlexNet, 93,199 GoogLeNet/Inception 93,95 ). Occasionally, these pretrained models are used in combination, as in the ensemble approach of Reference 200: this system leverages pretrained Inception-V3, 198 Xception, 201   and ResNet-50 77 as individual networks in a weighted voting scheme for binary classification of histopathological slides. Another study 202 provides a comparative analysis of different ImageNet-pretrained models within the context of colorectal cancer histopathology slide segmentation, finding that a DenseNet-121 203 feature extractor, paired with a LinkNet 204 segmentation architecture, is the most promising approach among the pretrained models they evaluated, which spanned DenseNets, Inception Networks, MobileNets, 205 ResNets, and VGG architectures.

| Transfer learning
Recent work 206 compares the performance of models pretrained on ImageNet with those pretrained using self-supervised (Section 3.3, Reference 207) or multitask learning 208 on histopathology data. On binary classification of slides as cancerous or noncancerous on a dataset containing 413 WSIs from duodenal biopsies, they find that the selfsupervised encoders achieve a greater AUC than those pretrained on ImageNet. Moreover, they observe no discernible relationship between a model's performance on ImageNet, and its subsequent performance on the downstream task. This presents a challenge for researchers and practitioners attempting to leverage transfer learning in practice, as these results suggest that there presently exists no pretraining heuristic that can accurately ascertain the performance of a fine-tuned ImageNet model on a downstream CPATH classification task.

| Self-supervised learning
Deep learning models are trained to maximize the accuracy of the model on a training set. This requires access to labels. Self-supervised learning refers to the class of methods that allow models to learn features of images relevant to a task without access to underlying labels.
The key insight that self-supervised learning leverages is that one can often use domain knowledge to create pseudo-labels or learning objectives which, when optimized, yield informative representations of images.

| Contrastive learning
Contrastive learning performs self-supervised learning by means of assigning pairs of instances in data to be "positive pairs" or "negative  223 The advantages of self-supervised learning appear to be most pronounced when a limited quantity of labeled data is available for domain-specific transfer learning.

| Neural attention with transformers
Attention is a type of neural network expressing the inductive bias that context determines the importance of each input variable to the current layer of the network. In computer vision, attention learns from surrounding pixels the importance of each pixel to the current layer of computation. Empirically, this inductive bias proves an effective assumption in the context of natural language and image processing.
Transformers, neural networks constructed using stacked neural attention layers, have set the new standard in natural language processing tasks, 189 while ViTs approach state-of-the-art results on vision tasks with significantly fewer parameters than convolutional networks. 218 One of the key strengths of the attention mechanism is interpretability: by visualizing the attention weights over each pixel as a heat map, a user can interpret the learned relative importance of each pixel to the ultimate prediction task (e.g., as in Reference 222).
This allows for domain experts to conduct post hoc interpretability of the learned model to assess whether the model has learned the right signal for the task at hand.
Much of the success of attention in CPATH has leveraged attention mechanisms as the pooling function in MIL. 224,225 In the former work, 224 use an attention mechanism as the pooling operator for MIL, instead of a fixed pooling function. In a classification task on breast 226 and colon cancer 116 data, their gated attention-based MIL approach achieved higher image-level binary classification accuracy, precision, recall, F-score, and AUC than either instance-wise or embedding-wise MIL approaches. In the latter work, 225

| Graph neural networks
GNNs are a class of neural networks that encode a relational inductive bias: the assumption that properties of-and relationships betweendiscrete entities in the data are important to the overall prediction task. 229 To do so, a GNN will compose each data sample as a series of nodes and edges and will learn a representation of this graph that most readily supports the overall prediction task. In the context of computational histopathology, nodes in the graph typically correspond to patches sampled from a slide. We can therefore group and compare GNN-based methods by the way in which the edges of each graph are assigned to their corresponding nodes.
3.5.1 | Node feature similarity Some methods will construct a graph out of each instance by placing an edge between nodes that are sufficiently similar to each other. This class of methods includes DeepGraphSurv, 156

| Node spatial location
In this paradigm, nodes in the GNN are typically represented by patches, while the graphical structure is produced based on which nodes are spatially proximal. This encodes the inductive bias that the presence of a feature in one location (e.g., a cancerous cell) is likely to inform the presence of that same feature in other nearby locations.
HGSurvNet 157 is a GNN-based method that performs survival prediction from WSIs. To do so, it constructs two hypergraphs, and performs multi-hypergraph learning over the two graphs to produce a downstream survival prediction. One of the hypergraphs contains edges determined by the feature similarity of nodes, as determined by feature extraction via an ImageNet-pretrained model, while the other contains edges determined by spatial proximity to other nodes on the slide. Training this architecture using the Cox partial likelihood objective 234 yields a survival model that outperforms competing methods like graph convolutional networks 48 and DeepGraphSurv to achieve a concordance of 0.6730 on the LUSC dataset, 235 0.6726 on the GBM dataset, 235 and 0.6901 on the NLST dataset. 230 Instead of using nuclei as nodes in the graph, 236 uses cell nuclei as the nodes, then connects nuclei in the graph that are sufficiently proximal. Applying attention-based robust spatial filtering on this graph yields near-stateof-the-art on subtype classification of breast cancer 86 and Gleason grading of prostate cancer 237 tasks and admits interpretable attention maps in which well-attended nuclei correlate strongly with the presence of a cancerous cell. Graphs based on node spatial location have also been used to improve the tractability of the learning problem: Slide Graph 238 constructs a graph in which nodes are cell nuclei, with edges placed between proximal nuclei. This approach provides a scalable means to efficiently capture cellular structure across a WSI. In evaluation on a dataset of breast cancer pathology slides from TCGA, this method achieved a 0.73 AUC on HER-2 status prediction (with the next closest baseline 124 achieving 0.68), and a 0.75 AUC on PR status prediction (with the next closest baseline 42 achieving 0.73).

| Patch spatial location with superpixel node features
Reference 239 presents SegGini, a graph isomorphism network that leverages superpixel node features to perform weakly-supervised semantic segmentation of histopathological slides. It does so by way of node classification, wherein superpixel nodes are each classified into segmentation regions. A key advantage of this method is its ability to operate under inexact labels and partial annotation, and in evaluation on one prostate tissue microarray dataset 240 and one prostate WSI dataset, 241 SegGini performs state-of-the-art segmentation as measured by the Dice score, outperforming a human clinician on the first dataset.  The naive training of ML models to maximize average accuracy on a predictive task has been found to yield models that are prone to bias among subpopulations within the data. This is because populations represented in real-world datasets are diverse and average accuracy on a held-out set may not reflect the nuances of how the model will performs on members of various subpopulations during deployment. 62,244 In predictive systems for chest x-rays, 245

| Heterogeneity of predictive outcomes
Digitized histopathology images can exhibit variation that is dependent on the tumor microenvironment, 251 the stage of the disease, 252 the stain used in the image, 253 and the patient's individual characteristics. 254 This results in intra-observer variability from the subjective and manual interpretation of these images by pathologists. The manual annotations of these images, in many cases, form the labels used to train CPATH models. Many ML models assume that the noise present in the labels has the same degree of variation; the violation of this assumption can exacerbate bias in the resulting model. Reference 255 studies the effect of instance dependent noise in neural network models, showing that low-frequency noisy labels (such as those coming from a minority subpopulation) are more likely to be misclassified.
A detailed study of the effects of label noise in computational histopathology, its origin, and mechanisms to mitigate its effects on generalization represents a promising area of future study and would further improve trust in CPATH models.

| Multimodal integration and the need for interpretability
The treatment and care of patients suffering from cancers involves clinical decision making from a variety of modalities. Integrating and harmonizing this data from electronic medical records and clinical trials opens new avenues for ML to ask novel research questions such as individual risk prognostication and biomarker identification. 256,257 For example, Reference 182 combines clinical biomarker data with histopathology images from the NRG Oncology phase III randomized clinical trials to predict outcomes such as metastases and survival in prostate cancer. 258  in the data when such relationships are statistically identifiable. [270][271][272] To our knowledge, these methods have yet to make their way into the computational histopathology literature. Such approaches may improve confidence that a model's interpretation is correctly characterizing the biological pathways linking WSI features and diagnostic outcomes, which-beyond improving trust in our predictive models-may improve our scientific understanding of the biological mechanisms relating observed WSI features with clinical outcomes.

| On the rise of large generative models
Discovery of scaling laws 273 for transformer-based models of natural language text has ushered in a new era of LLMs. Models in natural language processing were bespoke with a single model being trained on a dataset to solve a specific task at hand. By scaling models to hundreds of billions of parameters, researchers found that LLMs exhibit the ability to solve different natural language tasks with little to no supervision. In parallel, large scale diffusion models 274 have democratized the generation of high-resolution image data using text-prompts single sentence alone. The ramifications of this technology are only just being explored in the context of medicine 275 but the next half decade will inevitably find their utility in CPATH.

| DISCUSSION
To change patient care, a good ML model alone is insufficient. Equally important is the smooth integration of the model into the clinical workflow-an endeavor that intersect computational histopathology with human computer interaction. Indeed, creating reliable software tools for pathologists and oncologists would require a rethink of how hospital infrastructure is organized. As hospitals and clinics move toward an entirely digitized pathology workflow there is an opportunity to create new assistive clinical decision support tools using computational histopathology. This will require the implementation of high-throughput storage for digitized histopathology slides, fast interoperability with hospital electronic medical record systems and (local or cloud-based) high-performance compute to run ML models in real-time.
In summary, the increasing digitization of pathology workflows alongside the rapid pace of advances in ML bears promise for accelerating scientific discovery and in the creation of assistive tools for oncologists across a variety of cancers. As the field moves from research to translation and deployment, there is a need to recognize the ultimate end-uses of predictive systems within the clinical workflow and translate the technical requirements a system must satisfy into research challenges. The clinical translation of these tools and technologies will require pathologists, oncologists, computer scientists, hospital administrators and regulatory agencies to collaborate and develop an environment where clinicians can utilize such tools safely and effectively.

ACKNOWLEDGMENTS
The authors thank Richard J. Chen for many helpful discussions, comments on the manuscript, and help framing the introduction.

FUNDING INFORMATION
This study was supported by the AI Chair Award, Canadian Institute for Advanced Research and the Health Systems Impact Fellowship, Canadian Institutes of Health Research.