The association between elevated peripheral eosinophil counts and clinical pathology was first noted at the turn of the century by Loeffler who described a characteristic form of endomyocardial fibrosis in association with blood eosinophilia (7). Subsequent case series, using persistent AEC > 1,500/μL as a defining criterion for HES, noted an association between extremely elevated AEC (white blood cell counts >100,000/μL) and poor prognosis (8, 9). Consistent with these findings, patients with PDGFRA-positive myeloid neoplasm, one of the most aggressive forms of HES, have higher AECs than patients with other clinical subtypes of HES and dramatic resolution of clinical manifestations following normalization of the AEC with imatinib therapy (10).

Despite these findings and the large body of circumstantial evidence from case reports, case series and clinical practice documenting an association between the resolution of clinical manifestations of HES and normalization of the AEC, assessment of the AEC as a surrogate marker of disease activity has not been studied directly in the context of clinical trials to date. That said, the efficacy of mepolizumab as a steroid-sparing agent was associated with reduction of AEC in two placebo-controlled, double-blind trials in HES [one in subjects with steroid-responsive HES (11) and the second in subjects with eosinophilic granulomatosis with polyangiitis (EGPA) (12)], suggesting that AEC is a useful marker of disease activity. The results of ongoing and recently completed trials (NCT02130882; NCT02101138) in HES using agents that selectively target eosinophils should provide additional support for the utility of the AEC as a biomarker of response.

Although tissue eosinophilia would seem to be a more specific indicator of disease activity in HES, the utility of eosinophil quantification in tissue biopsies to monitor disease activity is hampered by the difficulty in obtaining samples, the patchy nature of eosinophilic tissue infiltration, and the fact that intact eosinophils may be absent despite clear evidence of their involvement by immunohistochemical staining for eosinophil granule proteins (EGPs) (13–16). To date, the best data associating tissue eosinophil numbers with clinical symptomatology come from EoE where the numbers of eosinophils in normal tissue have been defined (17), and suppression of tissue eosinophil counts has been associated with improved long-term prognosis (18). Unfortunately, despite encouraging data from a small open-label trial (19), randomized placebo-controlled trials using anti-IL-5 antibody therapy (mepolizumab and reslizumab) have not demonstrated an association between reduction in tissue eosinophilia and improved symptoms (20–22). Potential explanations for the lack of symptomatic improvement include incomplete depletion of tissue eosinophilia, involvement of other cell types and/or structural changes due to fibrosis and remodeling that may require a longer time frame for resolution.

Released during eosinophil activation and deposited in tissue in sites of eosinophilic inflammation, EGPs are attractive candidate biomarkers for the monitoring of disease activity in HES (23). They can be detected and quantified in the blood (24–26), body fluids (27), and tissue (13–15, 28–31) using various immunoassays, and blood and/or body fluid levels have been shown to correlate with tissue deposition of EGP in a wide range of HES, including EoE in the absence of peripheral eosinophilia (24).

There are a several biologically relevant differences between EDN, EPO, MBP, and ECP. MBP is the predominant protein in the core of the eosinophil secondary granule. It exists as two highly basic homologs, MBP-1 and MBP-2 (26), both of which circulate as neutral pH pro-proteins. Of note, most immunoassays do not distinguish between pro-MBP and MBP. Whereas EPO is quite specific for the eosinophil lineage, MBP-1, EDN, and ECP are also present in neutrophils and/or basophils albeit at lower levels (32, 33). This does not appear to affect their ability to be used as a proxy for eosinophil-associated tissue pathology in most settings but deserves mention.

Immunohistochemical staining of tissue for EGP has been extremely useful in clarifying the role of eosinophils in the pathogenesis of HES when intact eosinophils are not detectable. Moreover, EGP staining has been shown to correlate with disease activity in some settings. For example, in one study, serial skin biopsies from patients with episodic angioedema with eosinophilia demonstrated EGP staining only when symptoms were present (15, 34). Unfortunately, the utility of EGP tissue staining as a biomarker of disease activity is limited by the need for serial tissue sampling. To address this issue in EoE, a number of novel and less invasive techniques have been developed. These include the esophageal string test (35) and the cytosponge (36). Both techniques involve swallowing a string (in the case of the cytosponge, this is attached to a gelatin capsule containing a mesh) from which EGP can be eluted and quantified. Good correlation between eluted EGP levels and immunohistochemical staining of matched biopsies has been confirmed for both techniques (37). Finally, ultrasound visualization of granule protein density using MBP-1 labeled-insulin particles has been demonstrated in ex vivo monkey esophagi and may ultimately provide a third non-invasive tool for the measurement of tissue EGP in EoE (38). The applicability of these or similar techniques to other tissues remains to be seen.

The utility of measuring circulating levels of EGPs to monitor disease activity in HES has been somewhat controversial in large part due to the lack of standardization of sample collection (eosinophil lysis could lead to falsely elevated levels) and differing assay parameters between studies. Nevertheless, there are some data to suggest that circulating EGP levels have value in the monitoring of disease activity in HES. An interesting observation in this regard has been the association of elevated EDN and EPO levels with clinical manifestations rather than peak eosinophil count in patients with episodic angioedema and eosinophilia (24). A similar association between clinical disease and elevated EDN levels was seen in a study comparing subjects with asymptomatic familial eosinophilia to subjects with active HES (39).

Data from clinical treatment trials have been more difficult to interpret. Whereas decreases in serum EDN levels were reported in subjects who received active drug in a placebo-controlled trial of mepolizumab in HES (11) and ECP levels decreased in response to mepolizumab therapy in three patients with HES and eosinophilic dermatitis (40), AEC also decreased in both studies making it difficult to assess the added benefit of measuring EGP levels. Moreover, in a recent study of non-invasive biomarkers of EoE, AEC, but not serum levels of ECP, was predictive of residual disease following topical steroid therapy (41).

Measurement of EGP in body fluids, such as urine, that do not normally contain eosinophils, has the theoretical advantage of eliminating false positive results due to eosinophil lysis. Although there are no studies examining urine levels of EGP in HES to date, several small studies in atopic dermatitis (42, 43) demonstrated a correlation between urine levels of ECP and clinical disease severity. By contrast, no such relationship was noted in children with asthma (44).

A wide variety of eosinophil surface markers are reported to be up- or downregulated on activated eosinophils (23, 45), Many of these, including IL-5Rα, CD69, and CD44, have been shown to have altered expression on eosinophils from patients with HES, but also in patients with HE_US (39). Despite this, there are little longitudinal data assessing changes in expression of these activation markers in response to therapy in patients with HES. In a single study in EoE, expression of activation markers on blood eosinophils was unchanged by topical steroid therapy, although the effect of therapy on esophageal eosinophilia was not reported (46).

Despite its clear role in the production, activation and regulation of eosinophils, IL-5 has been disappointing as a biomarker of disease activity in HES. Although IL-5 levels correlate with AEC overall, serum IL-5 is undetectable in some patients with untreated HES [Figure 1, unpublished data from Ref. (47)]. In this regard, serum IL-5 levels do not contribute additional information when the AEC is known. In addition, increased serum IL-5 levels have been reported in the setting of clinical and hematologic remission following administration of several different biologics designed to target eosinophils, including mepolizumab and benralizumab (48, 49). The reasons for this are likely multifactorial and include measurement of IL-5/anti-IL-5 immune complexes (mepolizumab) and antibody blocking of IL-5 binding to its receptor (benralizumab). Soluble IL-5R is measurable in the serum of most, if not all, patients with HES, and levels are correlated with serum IL-5 levels (47). Whether this would provide a better biomarker of disease activity, owing to its reliable detection in serum in contrast to IL-5, remains to be seen. Finally, a number of studies have looked at other serum cytokines and chemokines as biomarkers of disease activity in HES. Of these, mediators of potential interest have been identified mostly in EGPA and include IL-25 (50), serum CCL17/thymus and activation-regulated chemokine (TARC) levels (51, 52), and CCL26/eotaxin-3 (53, 54). Interestingly, despite tissue data implicating CCL26/eotaxin-3 in the pathogenesis of EoE, serum levels of these mediators were not increased in EoE patients and were not altered by therapy (55). Finally, although some authors have reported elevated IL-3 in the plasma of patients with eosinophilia in conjunction with intracellular staining in CD8+ T cells (56), IL-3 is not universally detected in serum (57) of patients with HES, and the role of IL-3 as a biomarker in HES remains to be explored.

An exciting advance in biomarker development has been the use of molecular profiling techniques to identify patterns of expression that can be used to follow disease activity. This approach has been used successfully in EoE, where patterns of gene expression in esophageal biopsies have led to the development of an EoE molecular diagnostic panel (EDP) (58, 59) which is further discussed in the accompanying review, and a microRNA signature (60) that correlate with disease activity and response to therapy.

LHES is defined by the presence of a clonal or aberrant phenotypic T cell that secretes type 2 cytokines driving the eosinophilia and elevated serum IgE levels seen in this clinical variant (62, 63). Whereas the most common aberrant immunophenotype is CD3−CD4+, various aberrant immunophenotypes have been described (64), and some patients have cytokine-secreting clonal T-cell populations despite an apparently normal immunophenotype. Dermatologic manifestations, including angioedema, nodules, eczematous dermatitis, and erythroderma, are common in patients with LHES (65), and aberrant T cells can often be detected in skin biopsies from affected areas (66). Patients with LHES are often glucocorticoid responsive but typically require moderately high doses (67). Consequently, glucocorticoid-sparing agents with effects on T cells, such as interferon-alpha and cyclosporine, are frequently used. Although LHES is considered a benign lymphoproliferative disorder, a small proportion of LHES patients eventually develop a lymphoid malignancy, often heralded by expansion of the aberrant clonal T-cell population (62, 68). Conversely, regression of the aberrant T-cell population can be seen in response to effective therapy (Figure 2). Elevations in serum CCL17/TARC are more frequent in patients with LHES (69, 70), but information is lacking on the use of CCL17/TARC levels to monitor disease activity.

Myeloid HES refers to the subgroup of patients with HES in the setting of a primary myeloid disorder. Although most of these patients have detectable molecular abnormalities (most commonly the fusion gene FIP1L1-PDGFRA), others have a similar clinical phenotype of unknown cause. Clinical and laboratory features associated with MHES include dysplastic eosinophils, anemia and/or thrombocytopenia, elevated serum tryptase and B12 levels, and bone marrow features suggestive of a myeloid neoplasm (10). Before the availability of imatinib (a tyrosine kinase inhibitor with activity against PDGFRA and PDGFRB), mortality rates in patients with MHES were extremely high, due primarily to endomyocardial fibrosis and thromboembolic events. Currently, remission rates on imatinib therapy approach 100% in patients with PDGFR-associated disease and up to 50% in patients with other forms of MHES.

Although the AEC normalizes with effective therapy in MHES and can be used to monitor disease activity, data from chronic myelogenous leukemia and drug interruption trials in PDGFRA-associated HES suggest that molecular monitoring is preferable when possible since molecular relapse may precede hematologic (and clinical) relapse by several months (71). This is particularly important in view of recent data demonstrating sustained remission after imatinib discontinuation in some patients (72–74).

Overlap HES includes single organ and/or defined disorders that are characterized by eosinophilia and eosinophil-associated pathogenesis, including eosinophilic gastrointestinal disorders and EGPA. These disorders have distinct clinical presentations and complications and, for this reason, are often approached differently than other forms of HES. Although potential biomarkers for these conditions include the previously discussed general markers of eosinophilia and eosinophil activation, additional disease-specific issues are discussed below.