Recent Advances in Pathology: the 2020 Annual Review Issue of The Journal of Pathology

This year's Annual Review Issue of The Journal of Pathology contains 18 invited reviews on current research areas in pathology. The subject areas reflect the broad range of topics covered by the journal and this year encompass the development and application of software in digital histopathology, implementation of biomarkers in pathology practice; genetics and epigenetics, and stromal influences in disease. The reviews are authored by experts in their field and provide comprehensive updates in the chosen areas, in which there has been considerable recent progress in our understanding of disease. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

EMILIN2 down‐modulates the Wnt signalling pathway and suppresses breast cancer cell growth and migration

EMILIN2 is an extracellular matrix (ECM) protein that exerts contradictory effects within the tumour microenvironment: it induces apoptosis in a number of tumour cells, but it also enhances tumour neo‐angiogenesis. In this study, we describe a new mechanism by which EMILIN2 attenuates tumour cell viability. Based on sequence homology with the cysteine‐rich domain (CRD) of the Frizzled receptors, we hypothesized that EMILIN2 could affect Wnt signalling activation and demonstrate direct interaction with the Wnt1 ligand. This physical binding leads to decreased LRP6 phosphorylation and to the down‐modulation of β‐catenin, TAZ and their target genes. As a consequence, EMILIN2 negatively affects the viability, migration and tumourigenic potential of MDA‐MB‐231 breast cancer cells in a number of two‐ and three‐dimensional in vitro assays. EMILIN2 does not modulate Wnt signalling downstream of the Wnt–Frizzled interaction, since it does not affect the activation of the pathway following treatment with the GSK3 inhibitors LiCl and CHIR99021. The interaction with Wnt1 and the subsequent biological effects require the presence of the EMI domain, as there is no effect with a deletion mutant lacking this domain. Moreover, in vivo experiments show that the ectopic expression of EMILIN2, as well as treatment with the recombinant protein, significantly reduce tumour growth and dissemination of cancer cells in nude mice. Accordingly, the tumour samples are characterized by a significant down‐regulation of the Wnt signalling pathway. Altogether, these findings provide further evidence of the complex regulations governed by EMILIN2 in the tumour microenvironment, and they identify a key extracellular regulator of the Wnt signalling pathway. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Epithelium: at the interface of innate and adaptive immune responses

Several diseases of the airways have a strong component of allergic inflammation in their cause, including allergic rhinitis, asthma, polypoid chronic rhinosinusitis, eosinophilic bronchitis, and others. Although the roles played by antigens and pathogens vary, these diseases have in common a pathology that includes marked activation of epithelial cells in the upper airways, the lower airways, or both. Substantial new evidence indicates an important role of epithelial cells as both mediators and regulators of innate immune responses and adaptive immune responses, as well as the transition from innate immunity to adaptive immunity. The purpose of this review is to discuss recent studies that bear on the molecular and cellular mechanisms by which epithelial cells help to shape the responses of dendritic cells, T cells, and B cells and inflammatory cell recruitment in the context of human disease. Evidence will be discussed that suggests that secreted products of epithelial cells and molecules expressed on their cell surfaces can profoundly influence both immunity and inflammation in the airways.     

What do we know about the inflammasome in humans?

The inflammasome complex is part of the innate immune system, which serves to protect the host against harm from pathogens and damaged cells. It is a term first proposed by Tschopp's group in 2002, with numerous original research articles and reviews published on the topic since. There have been many types of inflammasome identified, but all result in the common pathway of activation of caspases and interleukin 1β along with possible cell death called pyroptosis. Despite a growing body of research investigating the structure and function of the inflammasome in animal models, there is still limited evidence identifying inflammasome components in human physiology and disease. In this review, we explore the molecular structure and mechanism of activation of the inflammasome with a particular focus on inflammasome complexes expressed in humans. Inflammasome components have been identified in several human peripheral and brain tissues using both in vivo and ex vivo work, and the inflammasome complex has been shown to be associated with several genetic and acquired inflammatory and neoplastic disorders. We discuss the strengths and weaknesses of the information available on the inflammasome with an emphasis on the importance of prioritizing work on human tissue. There is a huge demand for more effective treatments for a number of inflammatory and neurodegenerative diseases. Modulation of the inflammasome has been proposed as a novel treatment for several of these diseases and there are currently clinical trials ongoing to test this theory.     

Expression of miR‐100 and miR‐138 as prognostic biomarkers in non‐muscle‐invasive bladder cancer

microRNA alterations are involved in bladder cancer tumorigenesis. The aim of the current study was to evaluate the potential role of miR‐100 and miR‐138 as prognostic biomarkers in Ta/T1 non‐muscle‐invasive bladder cancer (NMIBC). We assessed a quantitative RT‐PCR analysis of miR‐100 and miR‐138 in 50 bladder tumor samples (stage Ta/T1) and four healthy adjacent tissues. Western blot analysis was used to measure protein expression of FGFR3 and cyclin D3 in order to know whether these targets can be regulated by miR‐100 and miR‐138, respectively. The statistical analysis included non‐parametric tests (Mann–Whitney U and Kruskal–Wallis) and univariate survival analysis by Kaplan–Meier method and the log‐rank test. Low expression of miR‐138 characterized recurrent tumors (p = 0.043), and higher expression levels were associated with longer recurrence‐free survival (p = 0.012). However, low miR‐100 expression correlated with longer progression‐free survival (marginal significance; p = 0.053) and cancer‐specific overall survival (p = 0.006). Additionally, higher levels of miR‐100 were associated with negative FGFR3 protein expression (p = 0.032) and higher levels of miR‐138 were associated with positive cyclin D3 protein expression (p = 0.037). Our results support miR‐138 and miR‐100 as prognostic biomarkers in patients with NMIBC.     

Reactive Oxygen Species in the Immune System

Reactive oxygen species (ROS) are a group of highly reactive chemicals containing oxygen produced either exogenously or endogenously. ROS are related to a wide variety of human disorders, such as chronic inflammation, age-related diseases and cancers. Besides, ROS are also essential for various biological functions, including cell survival, cell growth, proliferation and differentiation, and immune response. At present there are a number of excellent publications including some reviews about functions of these molecules either in normal cell biology or in pathophysiology. In this work, we reviewed available information and recent advances about ROS in the main immune cell types and gave summary about functions of these highly reactive molecules both in innate immunity as conservative defense mechanisms and in essential immune cells involved in adaptive immunity, and particularly in immune suppression.     

Stem cells in breast epithelia

The rodent and human nonpregnant mammary glands contain epithelial, intermediate and myoepithelial cells which have all been isolated as cell lines in vitro. Transforming growth factor-α (TGFα) and basic fibroblast growth factor (bFGF) are produced by myoepithelial cells and can stimulate the growth of intermediate stem cells in vitro. Epithelial and intermediate cells behave like stem cells in vitro, since they can differentiate into alveolar-like and myoepithelial cells. The myoepithelial differentiation pathway is associated with the early expression of a calcium-binding regulatory protein called p9Ka and the protease, Cathepsin D. Myoepithelial cells are also present in benign lesions but not in malignant mammary carcinomas of rats or humans, whose resultant cell lines fail to differentiate completely along the myoepithelial cell pathway. Loss of the myoepithelial cell in some invasive carcinomas may be compensated, at least in part, by changes in malignant cells. Over-expression of TGFα and/or erbB receptors may reduce the requirement for TGFα, whilst ectopic production of bFGF and its receptors and p9Ka/Cathespin D may assist in tumorigenesis and in metastasis, respectively. Thus compensation for, or retention of, molecules potentially involved in the differentiation of mammary cells may be a mechanism by which malignancy progresses in some human invasive carcinomas.     

NK cells – Versatile tools for viral defense and cancer treatment

About 150 NK cell researchers met at the German Cancer Research Center (DKFZ) in Heidelberg, Germany from 26–28 September 2012 for the Natural Killer Cell Symposium which was organized by the NK cell study group of the German Society for Immunology (DGfI) and sponsored by the European Journal of Immunology (EJI), the European Federation of Immunological Societies (EFIS) and the DGfI. The meeting was a forum for the discussion of the function and regulation of these fascinating innate immune cells and the opportunities for the transfer of this knowledge to cancer immunotherapy.     

Prevention and treatment of COVID-19 disease by controlled modulation of innate immunity

The recent outbreak of coronavirus disease 2019 (COVID-19), triggered by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses an enormous threat to global public health and economies. Human coronaviruses normally cause no or mild respiratory disease but in the past two decades, potentially fatal coronavirus infections have emerged, causing respiratory tract illnesses such as pneumonia and bronchitis. These include severe acute respiratory syndrome coronavirus (SARS-CoV), followed by the Middle East respiratory syndrome coronavirus (MERS-CoV), and recently the SARS-CoV-2 coronavirus outbreak that emerged in Wuhan, China, in December 2019. Currently, most COVID-19 patients receive traditional supportive care including breathing assistance. To halt the ongoing spread of the pandemic SARS-CoV-2 coronavirus and rescue individual patients, established drugs and new therapies are under evaluation. Since it will be some time until a safe and effective vaccine will be available, the immediate priority is to harness innate immunity to accelerate early antiviral immune responses. Second, since excessive inflammation is a major cause of pathology, targeted anti-inflammatory responses are being evaluated to reduce inflammation-induced damage to the respiratory tract and cytokine storms. Here, we highlight prominent immunotherapies at various stages of development that aim for augmented anti-coronavirus immunity and reduction of pathological inflammation.     

Mouse Models of Intracranial Aneurysm

Subarachnoid hemorrhage secondary to rupture of an intracranial aneurysm is a highly lethal medical condition. Current management strategies for unruptured intracranial aneurysms involve radiological surveillance and neurosurgical or endovascular interventions. There is no pharmacological treatment available to decrease the risk of aneurysm rupture and subsequent subarachnoid hemorrhage. There is growing interest in the pathogenesis of intracranial aneurysm focused on the development of drug therapies to decrease the incidence of aneurysm rupture. The study of rodent models of intracranial aneurysms has the potential to improve our understanding of intracranial aneurysm development and progression. This review summarizes current mouse models of intact and ruptured intracranial aneurysms and discusses the relevance of these models to human intracranial aneurysms. The article also reviews the importance of these models in investigating the molecular mechanisms involved in the disease. Finally, potential pharmaceutical targets for intracranial aneurysm suggested by previous studies are discussed. Examples of potential drug targets include matrix metalloproteinases, stromal cell‐derived factor‐1, tumor necrosis factor‐α, the renin‐angiotensin system and the β‐estrogen receptor. An agreed clear, precise and reproducible definition of what constitutes an aneurysm in the models would assist in their use to better understand the pathology of intracranial aneurysm and applying findings to patients.     

Pathology and Pathogenesis of Ovine Pulmonary Adenocarcinoma

Ovine pulmonary adenocarcinoma (OPA), also known as jaagsiekte, is a transmissible lung tumour of sheep caused by jaagsiekte sheep retrovirus (JSRV). JSRV induces neoplastic transformation of alveolar and bronchiolar secretory epithelial cells and the resulting tumours can grow to occupy a significant portion of the lung. Tumour growth is frequently accompanied by the overproduction of fluid in the lung, which further compromises normal respiration. The period between infection and the appearance of clinical signs may be several months or years and many JSRV-infected sheep do not exhibit clinical signs at all during their lifespan. This allows the spread of OPA into new flocks through contact with infected but apparently normal animals. OPA was first described in the early 19th century; however, it has still not been possible to devise effective methods for controlling its spread and it remains an important problem in most countries where sheep are farmed. This is due in part to the absence of an immunological response to JSRV in infected animals, which has hindered the development of serological diagnostic tests and vaccines. In addition to its veterinary importance, OPA is regarded as a potential large animal model for human lung adenocarcinoma and this has stimulated research into the pathogenesis of the ovine disease. This work has produced some significant results, including the finding that one of the JSRV structural proteins is directly involved in oncogenesis. The recent advances in understanding JSRV and the pathogenesis of OPA should lead to novel strategies for diagnosis and control of this disease and for its exploitation as a comparative model for human lung cancer.     

The collagen landscape in cancer: profiling collagens in tumors and in circulation reveals novel markers of cancer-associated fibroblast subtypes

Cancer-associated fibroblasts (CAFs) deposit and remodel collagens in the tumor stroma, impacting cancer progression and efficacy of interventions. CAFs are the focus of new therapeutics with the aim of normalizing the tumor microenvironment. To do this, a better understanding of CAF heterogeneity and collagen composition in cancer is needed. In this study, we sought to profile the expression of collagens at multiple levels with the goal of identifying cancer biomarkers. We investigated the collagen expression pattern in various cell types and CAF subtypes in a publicly available single-cell RNA sequencing (RNA-seq) dataset of pancreatic ductal adenocarcinoma. Next, we investigated the collagen expression profile in tumor samples across cancer types from The Cancer Genome Atlas (TCGA) database and evaluated if specific patterns of collagen expression were associated with prognosis. Finally, we profiled circulating collagen peptides using a panel of immunoassays to measure collagen fragments in the serum of cancer patients. We found that pancreatic stellate cells and fibroblasts were the primary producers of collagens in the pancreas. COL1A1, COL3A1, COL5A1, COL6A1 were expressed in all CAF subtypes, whereas COL8A1, COL10A1, COL11A1, COL12A1 were specific to myofibroblast CAFs (myCAF) and COL14A1 specific to inflammatory CAFs (iCAF). In TCGA database, myCAF collagens COL10A1 and COL11A1 were elevated across solid tumor types, and multiple associations between high expression and worse survival were found. Finally, circulating collagen biomarkers were elevated in the serum of patients with cancer relative to healthy controls with COL11A1 (myCAF) having the best diagnostic accuracy of the markers measured. In conclusion, CAFs express a noncanonical collagen profile with specific collagen subtypes associated with iCAFs and myCAFs in PDAC. These collagens are deregulated at the cellular, tumor, and systemic levels across different solid tumors and associate with survival. These findings could lead to new discoveries such as novel biomarkers and therapeutic targets. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.     

Controlled infection with a therapeutic virus defines the activation kinetics of human natural killer cells in vivo

Human natural killer (NK) cells play an important role in anti‐viral immunity. However, studying their activation kinetics during infection is highly problematic. A clinical trial of a therapeutic virus provided an opportunity to study human NK cell activation in vivo in a controlled manner. Ten colorectal cancer patients with liver metastases received between one and five doses of oncolytic reovirus prior to surgical resection of their tumour. NK cell surface expression of the interferon‐inducible molecules CD69 and tetherin peaked 24–48 h post‐infection, coincident with a peak of interferon‐induced gene expression. The interferon response and NK cell activation were transient, declining by 96 h post‐infection. Furthermore, neither NK cell activation nor the interferon response were sustained in patients undergoing multiple rounds of virus treatment. These results show that reovirus modulates human NK cell activity in vivo and suggest that this may contribute to any therapeutic effect of this oncolytic virus. Detection of a single, transient peak of activation, despite multiple treatment rounds, has implications for the design of reovirus‐based therapy. Furthermore, our results suggest the existence of a post‐infection refractory period when the interferon response and NK cell activation are blunted. This refractory period has been observed previously in animal models and may underlie the enhanced susceptibility to secondary infections that is seen following viral infection.     

Normal breast stem cells, malignant breast stem cells, and the perinatal origin of breast cancer

Both experimental and epidemiological evidence support the concept that the in utero environment can influence an individual's risk of breast cancer in adult life. Recently identified breast stem cells may be the key to understanding the mechanism underlying this phenomenon. It has been theorized that breast cancers arise from breast stem cells. Our emerging view of the characteristics of normal breast stem cells and their link to malignant breast stem cells is reviewed here. It has also been postulated that factors that expand the normal breast stem cell pool in utero would increase the probability that one such cell might undergo an oncogenic mutation or epigenetic change. We dicuss how a number of proposed perinatal determinants of adult breast cancer risk, including (1) in utero estrogen and IGF-1 levels, (2) birthweight, (3) breast density, and (4) early-life mutagen exposure, can be tied together by this “breast stem cell burden” hypothesis.     

Intracellular sensors of extracellular bacteria

Initial recognition of bacteria by the innate immune system is thought to occur primarily by germline-encoded pattern recognition receptors (PRRs). These receptors are present in multiple compartments of host cells and are thus capable of surveying both the intracellular and extracellular milieu for bacteria. It has generally been presumed that the cellular location of these receptors dictates what type of bacteria they respond to: extracellular bacteria being recognized by cell surface receptors, such as certain Toll-like receptors, and bacteria that are capable of breaching the plasma membrane and entering the cytoplasm, being sensed by cytoplasmic receptors, including the Nod-like receptors (NLRs). Increasingly, it is becoming apparent that this is a false dichotomy and that extracellular bacteria can be sensed by cytoplasmic PRRs and this is crucial for controlling the levels of these bacteria. In this review, we discuss the role of two NLRs, Nod1 and Nod2, in the recognition of and response to extracellular bacteria.