Proposed refined diagnostic criteria and classification of eosinophil disorders and related syndromes.

Eosinophilia and eosinophil activation are recurrent features in various reactive states and certain hematologic malignancies. In patients with hypereosinophilia (HE), HE-induced organ damage is often encountered and may lead to the diagnosis of a hypereosinophilic syndrome (HES). A number of known mechanisms and etiologies contribute to the development of HE and HES. Based on these etiologies and the origin of eosinophils, HE and HES are divided into primary forms where eosinophils are clonal cells, reactive forms where an underlying reactive or neoplastic condition is detected and eosinophils are considered to be "non-clonal" cells, and idiopathic HE and HES in which neither a clonal nor a reactive underlying pathology is detected. Since 2012, this classification and the related criteria have been widely accepted and regarded as standard. However, during the past few years, new developments in the field and an increasing number of markers and targets have created a need to update these criteria and the classification of HE and HES. To address this challenge, a Working Conference on eosinophil disorders was organized in 2021. In this conference, a panel of experts representing the relevant fields, including allergy, dermatology, hematology, immunology, laboratory medicine, and pathology, met and discussed new markers and concepts as well as refinements in definitions, criteria and classifications of HE and HES. The outcomes of this conference are presented in this article and should assist in the diagnosis and management of patients with HE and HES in daily practice and in the preparation and conduct of clinical trials.

Measurement of Inflammation in Eosinophilic Esophagitis Using an Eosinophil Peroxidase Assay.

OBJECTIVES: We describe a simple, quick method to measure an eosinophil granule protein, eosinophil peroxidase (EPO), as a marker of eosinophil activity, in eosinophilic esophagitis (EoE). METHODS: Esophageal mucosal brushings initially were collected from 36 patients with active EoE (n=13), resolved EoE (n=13), and controls (n=10) before endoscopic biopsy collection; the brushes were frozen at -80 ºC until assayed. EPO on the brush was measured in a colorimetric assay visually and by spectrophotometric absorbance measurements (at 492 nm), and was compared with peak eosinophil counts in esophageal biopsy specimens. The assay was calibrated with known EPO concentrations; as EPO increased in the assay, the color changed from light yellow to dark brown. RESULTS: Mucosal brush specimens from active EoE yielded orange to dark brown colors with absorbance measurements > 1.1 U; in contrast, control and resolved EoE brush specimens yielded a light to dark yellow color with absorbance measurements < 1.1. We then corroborated the results at the bedside (real time) in 16 additional patients. EPO on the brush was measured directly in < 1 h in the assay visually and by absorbance at 492 nm. Absorbance units strongly correlated with peak eosinophil counts both with the frozen brush (rs=0.79, P<0.0001) and with the bedside (rs=0.86, P<0.00017) approaches. CONCLUSIONS: The results support the use of this rapid method to detect and monitor EoE disease activity. Moreover, because eosinophils infiltrate and degranulate in the esophagus in EoE in a patchy manner, this method may be more accurate than current practice by testing for an eosinophil constituent from both intact and degranulated cells, and by sampling large portions of the esophageal lumen rather than small biopsy specimens that may not be representative of eosinophil involvement.

Extracellular Eosinophil Granule Protein Deposition in Ringed Esophagus with Sparse Eosinophils.

BACKGROUND: Eosinophilic esophagitis (EoE) remains difficult to classify because of varying presentations. Not uncommonly, patients present with symptoms of esophageal dysfunction and have esophageal changes on endoscopy resembling EoE but without >15 eosinophils/HPF. Patients with low numbers of eosinophils in esophageal biopsy specimens may have esophageal changes and symptomatic disease brought about by eosinophil granule protein deposition without recognizable intact cells. AIM: To determine whether extracellular eosinophil granule protein deposition is present in the esophagi of patients with low eosinophil numbers who have clinical symptoms and characteristic endoscopic esophageal changes of EoE including ringed esophagus (RE). METHODS: Esophageal biopsy specimens were studied from eight EoE patients with >15 eosinophils per high power field (HPF) and nine patients with RE (<15 eosinophils/HPF). The specimens were analyzed for eosinophil granule proteins, major basic protein 1 (eMBP1) and eosinophil-derived neurotoxin (EDN), by indirect immunofluorescence. RESULTS: Both EoE and RE showed positive EDN and eMBP1 extracellular deposition; control esophagus showed minimal or none. Comparing EoE and RE, extracellular EDN and eMBP1 were similar except that EDN in EoE was greater in the distal esophagus. CONCLUSIONS: This study highlights the importance of assessing eosinophil granule protein deposition in esophageal disease with potential eosinophil involvement. Persistent/progressive esophageal changes may be brought about by eosinophil granule proteins despite low numbers of intact cells. The meaning of "resolution" in EoE may need to be redefined based on numbers of esophageal eosinophils, extracellular eosinophil granule protein deposition, and subsequent clinical course of patients.

Electron microscopy elucidates eosinophil degranulation patterns in patients with eosinophilic esophagitis.

BACKGROUND: In patients with eosinophilic esophagitis (EoE), eosinophils accumulate and release granule proteins onto esophageal epithelium. However, little is understood about the mechanism of eosinophil degranulation. OBJECTIVE: To determine and quantify eosinophil degranulation patterns, we studied esophageal biopsy specimens from both the proximal and distal esophagi of 9 randomly selected patients with EoE. METHODS: The specimens were fixed in glutaraldehyde, embedded, sectioned, and imaged by means of transmission electron microscopy. Eosinophils and their granules were identified by their distinctive morphology, and all eosinophils and granules were imaged. A total of 1672 images from 18 esophageal specimens were evaluated and graded. Eosinophils were categorized based on membrane integrity and by cytoplasmic vesiculation as evidence of piecemeal degranulation. Granules were categorized based on reversal of staining (eosinophil granule core lightening) and localization within and outside the cells. RESULTS: The results revealed that greater than 98% of eosinophils infiltrating the esophagus in patients with EoE demonstrate morphologic abnormalities ranging from granule changes with reversal of staining to marked cytoplasmic vesiculation to loss of cellular membrane integrity with cytolytic disruption and release of intact membrane-bound granules into the tissues. Approximately 81% of eosinophils showed membrane disruption. Extracellular granules were abundant in at least 70% of the images, and approximately 50% of these granules showed reversal of staining. On the basis of the prominence of tubulovesicular development, piecemeal degranulation appears closely related to the other morphologic changes seen in patients with EoE. CONCLUSION: These findings reveal that eosinophils in esophageal biopsy specimens from patients with EoE are abnormal, with greater than 80% showing cytolysis, and therefore that evaluation by means of light microscopy after hematoxylin and eosin staining might not accurately reflect eosinophil involvement.

The true extent of eosinophil involvement in disease is unrecognized: the secret life of dead eosinophils.

Eosinophil-mediated pathophysiology is tissue destructive and tissue altering with proinflammatory, prothrombotic, and profibrotic effects. The distinctive morphology of an eosinophil reveals a cytoplasm chockfull of unique granules, and the granule proteins have numerous toxic effects on cells, tissues, and organs. Eosinophils are not found in most human tissues, and eosinophil involvement in diseased tissues generally is identified by cell infiltration on histopathologic examination. However, eosinophils characteristically lose their structural integrity and deposit granules and granule proteins at sites of inflammation. Hence, their participation in tissue damage may be underrecognized or entirely overlooked. The eosinophil major basic protein 1 is a toxic granule protein and, when deposited, persists in tissues. Major basic protein 1 deposition can be regarded as a footprint of eosinophil activity. Analyses of numerous eosinophil-related diseases have demonstrated clear-cut evidence of major basic protein 1 deposition in affected tissues where eosinophils were not recognized by hematoxylin and eosin tissue staining and light microscopy. Eosinophil granule protein deposition, as exemplified by localization of major basic protein 1, especially when disproportionately greater than cellular infiltration, emerges as a biomarker of hidden eosinophil-related pathophysiology. Consequently, current assessments of recognized eosinophils may vastly underestimate their role in disease.

Contemporary consensus proposal on criteria and classification of eosinophilic disorders and related syndromes.

Eosinophilia is an important indicator of various neoplastic and nonneoplastic conditions. Depending on the underlying disease and mechanisms, eosinophil infiltration can lead to organ dysfunction, clinical symptoms, or both. During the past 2 decades, several different classifications of eosinophilic disorders and related syndromes have been proposed in various fields of medicine. Although criteria and definitions are, in part, overlapping, no global consensus has been presented to date. The Year 2011 Working Conference on Eosinophil Disorders and Syndromes was organized to update and refine the criteria and definitions for eosinophilic disorders and to merge prior classifications in a contemporary multidisciplinary schema. A panel of experts from the fields of immunology, allergy, hematology, and pathology contributed to this project. The expert group agreed on unifying terminologies and criteria and a classification that delineates various forms of hypereosinophilia, including primary and secondary variants based on specific hematologic and immunologic conditions, and various forms of the hypereosinophilic syndrome. For patients in whom no underlying disease or hypereosinophilic syndrome is found, the term hypereosinophilia of undetermined significance is introduced. The proposed novel criteria, definitions, and terminologies should assist in daily practice, as well as in the preparation and conduct of clinical trials.

Workshop report from the National Institutes of Health Taskforce on the Research Needs of Eosinophil-Associated Diseases (TREAD).

BACKGROUND: Eosinophils are blood cells that are often found in high numbers in the tissues of allergic conditions and helminthic parasite infections. The pathophysiologic roles that eosinophils may serve in other human "eosinophil-associated" diseases remain obscure. OBJECTIVE: National Institutes of Health (NIH) Institutes and the Office of Disease Prevention assembled an international taskforce of clinical and basic scientists with the charge to propose and prioritize unmet research needs in eosinophil-associated diseases. METHODS: The taskforce used an organ system approach to identify the different and common themes of eosinophil cell involvement in these diseases. In early 2012, a draft document was circulated for review. The document was amended and the prioritizations were set at a NIH-organized workshop in June 2012. RESULTS: The taskforce identified significant research needs. These needs cross disease entities but some are disease specific. There are substantial shortcomings to the various preclinical animal models, as well as significant gaps in our epidemiologic, pathophysiologic, diagnostic, prognostic, and therapeutic knowledge. The taskforce recognized that recent efforts by patient advocacy groups have played instrumental roles in improving the identification and characterization of these disorders. However, communications among the eosinophil-interested communities, for example, governmental funding and regulatory agencies, and industry and clinician scientists need to be more comprehensive. CONCLUSIONS: Significant efforts are required to address our knowledge gaps to improve the outcomes of eosinophil-associated diseases. NIH Institutes, other federal agencies, lay organizations, and the pharmaceutical industry should consider the taskforce's recommendations in their future research activities.

Diagnostics for Dermatologic Diseases with Autoantibodies.

BACKGROUND: Dermatologic diseases with autoantibodies were recognized early as autoimmunity became accepted as a pathogenic immunologic concept. Laboratory testing to identify disease-defining autoantibodies and investigate their role in pathophysiology has evolved since. CONTENT: Blistering dermatologic diseases, profiled by autoantibody production, target epithelial components critical in cell-cell and cell-matrix adhesion, resulting in epithelial separation and other characteristic features of the disorders. This review covers the clinical indications for dermatologic disease-related autoantibody testing, the specifics of procuring specimens to test, the available diagnostic tests, and information provided by the testing. Atypical, uncharacteristic, and less well-known clinical and autoantibody profiles as well as several of the many future prospects for expansion of the testing applications are elaborated on in the online Data Supplement. SUMMARY: Autoantibody-associated dermatologic diseases are acquired immunologic disorders that have considerable clinical implications affecting essential barrier functions of skin and mucous membranes and causing discomfort, including pain and pruritus. Certain of the diseases can have life-threatening manifestations, and treatments can have significant side-effects. The skin diseases may presage other clinical associations that are important to recognize and treat. Laboratory testing aids in the diagnosis of these diseases through identification of the autoantibodies and is essential for prompt and precise knowledge of the disease type for prognosis, further clinical evaluations, and treatment decisions.

Autoimmunity to desmocollin 3 in pemphigus vulgaris.

Pemphigus vulgaris is a blistering disease associated with autoantibodies to the desmosomal adhesion protein, desmoglein 3. Genetic deficiency of desmoglein 3 in mice mimics autoimmunity to desmoglein 3 in pemphigus vulgaris, with mucosal-dominant blistering in the suprabasal layer of the epidermis. Mice with an epidermal-specific deletion of desmocollin 3, the other major desmosomal cadherin isoform expressed in the basal epidermis, develop suprabasal blisters in skin that are histologically identical to those observed in pemphigus vulgaris, suggesting that desmocollin 3 might be a target of autoantibodies in some pemphigus vulgaris patients. We now demonstrate that desmocollin 3 is an autoantigen in pemphigus vulgaris, illustrated in a patient with mucosal-dominant blistering. Six of 38 pemphigus vulgaris and one of 85 normal serum samples immunoprecipitate desmocollin 3 (P = 0.003). Incubation of patient IgG with human keratinocytes causes loss of intercellular adhesion, and adsorption with recombinant desmocollin 3 specifically prevents this pathogenic effect. Additionally, anti-desmocollin 3 sera cause loss of keratinocyte cell surface desmocollin 3, but not desmoglein 3 by immunofluorescence, indicating distinct cellular pathogenic effects in anti-desmocollin and anti-desmoglein pemphigus, despite their identical clinical presentations. These data demonstrate that desmocollin 3 is a pathogenic autoantigen in pemphigus vulgaris and suggest that pemphigus vulgaris is a histological reaction pattern that may result from autoimmunity to desmoglein 3, desmocollin 3, or both desmosomal cadherins.

Eosinophil ribonucleases and their cutaneous lesion-forming activity.

Eosinophil granule proteins are deposited in cutaneous lesions in many human diseases, but how these proteins contribute to pathophysiology is obscure. We injected eosinophil cationic protein (ECP or RNase 3), eosinophil-derived neurotoxin (EDN or RNase 2), eosinophil peroxidase (EPO), and major basic protein-1 (MBP1) intradermally into guinea pig and rabbit skin. ECP and EDN each induced distinct skin lesions at >or=2.5 microM that began at 2 days, peaking at approximately 7 days and persisting up to 6 wk. These lesions were ulcerated (ECP) or crusted (EDN) with marked cellular infiltration. EPO and MBP1 (10 microM) each produced perceptible induration and erythema with moderate cellular infiltration resolving within 2 wk. ECP and EDN localized to dermal cells within 2 days, whereas EPO and MBP1 remained extracellular. Overall, cellular localization and RNase activity of ECP and EDN were critical for lesion formation; differential glycosylation, net cationic charge, or RNase activity alone did not account for lesion formation. Ulcerated lesions from patients with the hypereosinophilic syndrome showed ECP and EDN deposition comparable to that in guinea pig skin. In conclusion, ECP and EDN disrupt skin integrity and cause inflammation. Their presence in ulcerative skin lesions may explain certain findings in human eosinophil-associated diseases.

Hypereosinophilic syndrome: a multicenter, retrospective analysis of clinical characteristics and response to therapy.

BACKGROUND: Hypereosinophilic syndrome (HES) is a heterogeneous group of rare disorders defined by persistent blood eosinophilia > or =1.5 x 10(9)/L, absence of a secondary cause, and evidence of eosinophil-associated pathology. With the exception of a recent multicenter trial of mepolizumab (anti-IL-5 mAb), published therapeutic experience has been restricted to case reports and small case series. OBJECTIVE: The purpose of the study was to collect and summarize baseline demographic, clinical, and laboratory characteristics in a large, diverse cohort of patients with HES and to review responses to treatment with conventional and novel therapies. METHODS: Clinical and laboratory data from 188 patients with HES, seen between January 2001 and December 2006 at 11 institutions in the United States and Europe, were collected retrospectively by chart review. RESULTS: Eighteen of 161 patients (11%) tested were Fip1-like 1-platelet-derived growth factor receptor alpha (FIP1L1-PDGFRA) mutation-positive, and 29 of 168 patients tested (17%) had a demonstrable aberrant or clonal T-cell population. Corticosteroid monotherapy induced complete or partial responses at 1 month in 85% (120/141) of patients with most remaining on maintenance doses (median, 10 mg prednisone equivalent daily for 2 months to 20 years). Hydroxyurea and IFN-alpha (used in 64 and 46 patients, respectively) were also effective, but their use was limited by toxicity. Imatinib (used in 68 patients) was more effective in patients with the FIP1L1-PDGFRA mutation (88%) than in those without (23%; P < .001). CONCLUSION: This study, the largest clinical analysis of patients with HES to date, not only provides useful information for clinicians but also should stimulate prospective trials to optimize treatment of HES.

Oral Administration of 99mTechnetium-Labeled Heparin in Eosinophilic Esophagitis.

OBJECTIVE: To determine if heparin labeled with 99mTechnetium (99mTc) could be an imaging probe to detect eosinophil-related inflammation in eosinophilic esophagitis and to determine the biodistribution and radiation dosimetry of 99mTc-heparin oral administration using image-based dosimetry models with esophageal modeling. METHODS: Freshly prepared 99mTc-heparin was administered orally to 5 research subjects. Radioactivity was measured by whole-body scintigraphy and single-photon emission computed tomography during the 24 hours postadministration. Following imaging, endoscopic examination was performed. The biodistribution of esophageal radioactivity was compared with endoscopic findings, eosinophil counts in biopsy tissues, and immunostaining for eosinophil granule major basic protein-1 (eMBP1). These studies were conducted from July 1, 2013, until April 22, 2017. RESULTS: Oral administration of 99mTc-heparin was well tolerated in all 5 subjects. The entire esophagus could be visualized dynamically during oral administration. Bound esophageal radioactivity marked areas of inflammation as judged by endoscopy scores, by eosinophils per high power field and by localization of eMBP1 using immunostaining. Ninety percent of the radioactivity did not bind to the esophagus and passed through the gastrointestinal tract. CONCLUSION: The biodistribution of ingested 99mTc-heparin is almost exclusively localized to the gastrointestinal tract. Radiation exposure was highest in the lower gastrointestinal tract and was comparable with other orally administered diagnostic radiopharmaceuticals. The use of swallowed 99mTc-heparin may aid in assessing eosinophil-related inflammation in the esophagus.

The hypereosinophilic syndromes: current concepts and treatments.

The hypereosinophilic syndromes (HES) encompass a spectrum of diseases that have increased blood eosinophils and tissue damage in common. The clinical manifestations are protean and may involve any organ system, but especially the skin. Our understanding of these diseases has drastically changed over the past 15 years, along with new classifications that characterize patients with marked eosinophilia. One HES variant, myeloproliferative, is actually chronic eosinophilic leukaemia with a unique genetic marker, FIP1L1-PDGFRA. Such patients are well-controlled by administration of the kinase inhibitor, imatinib, and remissions appear durable with continued imatinib therapy. FIP1L1-PDGFRA is expressed in several cell lineages, thus explaining increases in neutrophils and mast cells in HES. The lymphocytic HES variant is associated with T-cell clones producing interleukin-5 (IL-5) and can evolve into lymphoma. While myeloproliferative and lymphocytic HES are well established and permit elimination of the term, idiopathic, to these varieties, most HES patients do not fall into these categories and are classified as complex (using the 2006 Workshop Report). A recent study showed that a monoclonal antibody to IL-5, mepolizumab, reduced glucocorticoid therapy in HES patients who did not possess the FIP1L1-PDGFRA mutation while controlling eosinophilia and preventing recurrence or progression of tissue damage. These advances augur well for continued progress in the understanding and treatment of HES.

Anaphylaxis: implications of monoclonal antibody use in oncology.

Anaphylaxis is currently classified as an immunologically triggered response with reactions that are IgE-mediated and reactions that are not IgE-mediated. This immunologically mediated phenomenon can result in various clinical manifestations, including decreased blood pressure, generalized skin inflammation, such as hives and pruritus, and respiratory symptoms, such as wheezing or bronchospasm. The severity of anaphylaxis can range from a mild allergic reaction to a potentially fatal anaphylactic shock. Numerous causative agents trigger anaphylactic reactions, and some of the best described include food and bee sting allergens. Monoclonal antibodies, which are increasingly used in the treatment of various malignancies, also can cause anaphylaxis. In this review, the mechanisms governing anaphylaxis along with treatment strategies are reviewed. Diagnostic aids for anaphylaxis are also discussed. Increased awareness of the mechanisms, symptoms, and treatment of anaphylaxis can aid caregivers to make informed decisions when new agents, such as monoclonal antibodies, are introduced into the clinic.

The hypereosinophilic syndromes: still more heterogeneity.

In 1968, the term hypereosinophilic syndromes (HES) was coined to refer to a spectrum of eosinophil-associated diseases presumed to be caused by an underlying immunological pathology. In the 1990s, the identification of an HES subset with T lymphocyte clonality and production of cytokines, particularly IL-5, validated this concept. Then, in 2002, imatinib mesylate, which was introduced for the treatment of chronic myelogenous leukemia, effectively controlled another subgroup of HES patients. Imatinib's target is a novel constitutively-active kinase. Most imatinib-responsive HES patients show an increased number of bone marrow mast cells and elevated serum tryptase; mast cells, lymphocytes and neutrophils express the novel kinase. This new information critically modifies our view of HES and indicates that several cell lines are altered and likely to contribute to HES pathophysiology.

Eosinophil-Related Disease and the Skin.

Eosinophils are bone marrow-derived cells that infiltrate skin and mucous membrane in a broad spectrum of primary and reactive inflammatory diseases and malignancies. The eosinophil has potent proinflammatory activities, particularly, through the effects of its toxic granule proteins. In addition, eosinophils have prothrombotic and profibrotic activities. Eosinophil participation in the pathogenesis of certain diseases without identifiable intact eosinophil infiltration may not be recognized because eosinophil degranulation is poorly visualized on hematoxylin-and-eosin-stained histopathology sections. Eosinophil-related pathophysiology can involve virtually every component of skin. Commonly recognized dermatoses associated with eosinophils are arthropod bite and sting reactions and drug eruptions, "bugs and drugs." Skin involvement is common in eosinophil-related systemic diseases including the hypereosinophilic syndromes. Eosinophil-related pathophysiology may play a key role in numerous disorders that, therefore, may benefit from therapies targeted to reduce or eliminate eosinophils.