Potential Biomarkers for Allergic Conjunctival Diseases

Abstract

Allergic conjunctival diseases (ACDs), are a group of ocular allergies that includes allergic conjunctivitis, atopic keratoconjunctivitis, vernal keratoconjunctivitis and giant papillary conjunctivitis. While a large body of information exist on the pathophysiology of ACDs, this has not yet lead to the development of clear recommendations and guidelines for the diagnosis of ACDs or development of conclusive and objective diagnostic tools. Identification of objectively measurable biomarkers that represent the molecular and cellular mechanisms associated with ACDs will be an important step towards achieving these aims. This is a comprehensive review of biological markers that have the potential to become ‘biomarker(s)’ for ACDs and aid in the classification, diagnosis and development of new therapeutic strategies for these group of allergic conditions.

Introduction

Allergic conjunctival Diseases (ACDs) are a group of conjunctival inflammatory diseases spanning different classes of hypersensitivity reactions and are accompanied by some subjective and objective symptoms¹ and affect approximately 10% to 20% of the US population². Importantly, ACDs occur as comorbidity of atopic diseases which affects 5% to 20% of the general population and therefore represents a major economic burden worldwide^3–5 . The diagnosis of ACDs are based on a combination of clinical history, signs and symptoms and wherever possible, in vivo and in vitro tests to identify specific allergens⁶. However, there are several complicating factors in the diagnosis of ACDs, the main one being the presence of clinical manifestations that overlap with other nonallergic ocular surface diseases like dry eye disease⁷, conjunctivitis associated with blepharitis, infections, amongst others⁸. Consequently, there is a lack of clear recommendations and guidelines for the diagnosis of ACDs, which is further compounded by the paucity of accurate and rapid diagnostics tools. Identification of biomarkers that represent molecular and cellular mechanisms associated with ACDs will be an important step towards achieving that. Objectively measurable biomarkers in combination with patient symptoms and currently available measures of signs may produce algorithms for diagnosis as well as provide direction towards developing new diagnostic tools and therapies for ACDs. This review will focus on existing insights into the pathophysiology of ACDs and the current status on potential biomarkers for ACDs.

Classification of ACDs

ACDs are classified into 4 main types based on signs, symptoms and the presence of atopic comorbidities.

1. Allergic conjunctivitis (AC).

This includes seasonal allergic conjunctivitis (SAC) where symptoms appear in a seasonal manner and perennial allergic conjunctivitis (PAC) where symptoms persist throughout the year. This group represents 25% to 50% of all cases of ACDs and is the most common allergic disease of the eye. SAC is associated with hypersensitivity to airborne allergens, such as tree and weed pollens or environmental antigens specific to a certain geographic area. PAC is associated with indoor antigens, such as dust mites, animal dander, and molds. Both SAC and PAC are primarily associated with IgE-mediated mast cell degranulation and consequent release of proinflammatory mediators and recruitment of eosinophils.

2. Atopic keratoconjunctivitis (AKC).

AKC is a severe chronic allergic conjunctival disease that requires prompt and effective treatment to prevent permanent vision loss. Complications of atopic keratoconjunctivitis include infectious keratitis, keratoconus, blepharitis, and cataracts. AKC is a comorbidity in 20% to 43% of individuals with atopic dermatitis (AD)^4,9. The immunopathology of AKC is complex, with both type I and IV hypersensitivity reactions contributing to the condition.

3. Vernal keratoconjunctivitis (VKC).

VKC represents 0.5% of allergic ocular disease¹⁰ and like AKC, many cases are associated with AD. VKC predominantly affects males, mainly children and young adolescents ages 11 to 13 years. Approximately 50% of patients have a history of atopy, such as AD, asthma and allergic rhinitis. Along with AKC, VKC it is primarily responsible for the blinding corneal complications of ACDs. Like AKC, both type I and IV hypersensitivity reactions contribute to the pathogenesis of VKC, with Th2 lymphocyte activation in association with high eosinophilic infiltration being predominant contributors to the pathophysiology.

4. Giant papillary conjunctivitis (GPC).

GPC is commonly present in contact lens wearers. Patients with history of atopy are at higher risk for GPC, as well as those exposed to materials such as ocular prostheses and sutures. Protein deposits on contact lenses or prostheses may serve as allergens creating a type I hypersensitivity or type IV hypersensitivity. The main underlying pathophysiology of GPS is Th2 lymphocyte mediated response to the mechanical trauma of the conjunctival epithelium.

Overview of the pathophysiology of ACDs

The conjunctiva is an immunologically primed barrier that consists mainly of an epithelial layer that is populated with goblet cells, lymphocytes and Langerhans cells and a subepithelial layer populated with a rich network of blood vessels, resident antigen presenting cells (APC) like dendritic (DCs) and mast cells^11,12. Collectively the components of the conjunctiva are equipped to mount a complex signaling cascade via their expression of toll-like receptors (TLR), histamine, histamine receptors, cytokines, chemokines, matrix metalloproteinases, and adhesion molecules^11,13–17. These together are some of the key mediators in the immunopathogenesis of ACDs, which is classically considered to have a sensitization phase, an early phase and a late phase.

Following the initial exposure of allergen specific IgE to conjunctival mast cells, the sensitization phase is initiated. Studies show that the initial breakdown of the epithelial layer cell junctions, mainly mediated by protease-activated receptor-2, exposes the allergens to DCs resulting in the subsequent differentiation of CD4 cells into allergen specific Th2 cells. This leads to the production of IL-4, differentiation of B-cells to plasma cells to produce allergen specific IgE and mast cell activation^18,19. Damaged epithelial cells produce IL-33, a potent Th2 cytokine that promotes IgE isotype switch and eosinophil infiltration. A study has shown the expression of IL-33 by epithelial and vascular endothelial cells in ACD patient conjunctival tissues^5,20. Additionally , IL-33 is a ligand for suppression of tumorigenity 2L (ST2L), and the ST2L gene has been found to be associated with a number of atopic conditions²⁰. Thymic stromal lymphopoietin (TSLP) is another cytokine that is produced by activated conjunctival epithelium through a TLR4-dependent pathway²¹. TSLP in turn can activate conjunctival DCs to express OX40 ligand and migrate to the regional lymph node to promote Th2 differentiation^22,23. Finally, Th2 cells along with conjunctival epithelial cells push conjunctival mast cells into the sensitization phase of allergic conjunctivitis²². While Th2 cells are considered to be the predominant mediators in ocular allergy, evidence exists for the involvement of Th1 and Th17 in severe chronic forms of AKC and VKC²⁴.

The early phase is primarily initiated by re-exposure and binding of allergens to the IgE–FcεRI complex on primed conjunctival mast cells^25,26. Mast cell degranulation leads to the release of histamine and neutral proteases like tryptase followed by release of lipid mediators that include prostaglandins and leukotrines. At this point a number of cytokines are produced, with IL-4 being the major one released^18,27. Studies have shown higher levels of IL-4 in the tears of patients with AKC, VKC, SAC compared to normal^28,29. Released histamine binds with H1 and H4 receptors on conjunctival epithelium, mast cells and Th2 cells to further perpetuate conjunctival inflammation^30,31. In this regards histamine H1 receptor antagonists are used for the treatment of SAC and PAC³². Also histamine and tryptase have been specifically shown to be associated with IgE-mediated mast cell activation and degranulation in the early phase of allergic conjunctivitis²⁷.

The late phase involves the downstream effect of mast cell degranulation and the released lipid mediators, including prostaglandins and leukotrienes both of which have been shown to potentiate the histamine-mediated vasodilation of conjunctival blood vessels, one of the major clinical signs of allergic conjunctivitis¹⁹. Cytokines and chemokines, including, IL-4, IL-5, IL-8 , IL-13 and CCL17, released from degranulated conjunctival mast cells initiate the recruitment of eosinophils into the late phase. IL-5 in particular has been shown to be important in the recruitment and activation of eosinophils in allergic conjunctivitis (AC), where it is involved in ocular surface remodeling, especially in PAC, AKC and VKC¹⁹. A study has shown that concentration of histamine in the tears of ACD patients reportedly increases during the late phase of immediate hypersensitivity³³. A study by Enriquez et al have demonstrated increased expression of IL-8 in the conjunctival epithelium of VKC and AKC patients and has been shown to be potent attractor of T-cells, eosinophils and neutrophils³⁴.

Putative Biomarkers for ACD

Studies with patient biological materials including tear, serum and tissue have yielded a number of potential biomarkers. Table 1 summarizes key studies that have aided in highlighting important potential biomarkers and Table 2 is a list of putative ‘biomarkers’ for ACDs that have immerged from these studies.

Table 1.

Summary of key studies providing insights into putative biomarkers for ACDs

Study	Purpose	Patient	Assay	Results/Conclusions
Matsuda et al, 2019108	Gene expression profiles of giant papillae tissues obtained from patients with AKC	n=3 AKC n=3 controls	RNA sequencing performed with Illumina HiSeq 1500	High number of immunoglobulin-encoded transcripts -suggesting that local immunoglobulin production is feature of AKC Genes within the S aureus infection pathway, ICAM1, complement-associated transcripts (C1QC, C1QB, and CFI), and formyl peptide receptors (FPR1 and FPR2), known to be associated with process host defense against S aureus Differentially expressed genes included TH2 cytokines, epithelial cell-derived type 2 initiate cytokines, and eosinophil-, macrophage-, and neutrophil-attracting chemokines Signature genes of mast cells (TPSAB1, CPA3, and FCER1A), eosinophils (PRG2, encoding eosinophil major basic protein), T cells(CD4), dendritic cells (ITGAX encoding CD11c), and Langerhans cells (CD207)
Shoji et al, 201850	To evaluate the relationship between clinical severity and tear levels of ECP and CCL23 in patients with chronic ACD	n=21 ACD n=14 controls	ELIAS for ECP and CCL23	In chronic ACD patients, tear ECP level significantly correlated with the clinical severity score. Clinical severity scores and tear ECP levels in the CCL23-positive subgroup were significantly higher than those in the CCL23-negative subgroup.
Andrade et al, 2018101	To evaluate Gal-3, a β-galactoside binding protein, as a possible biomarker in ocular allergy	n=29 ACD chronic n=3 controls	Immunostaining for Gal-3 in impression cytology samples	Higher levels of Gal-3 in ACD chronic vs controls
Shoji et al , 201739	Clinical usefulness of simultaneously determination of tear levels of CCL17/TARC, CCL24/eotaxin-2, and IL-16 for assessing acute and chronic allergic inflammation in ACDs	n=17 AC n= 6 AKC n=14 VKC n=11 controls	Magnetic bead assay CCL17/TARC, CCL24/eotaxin-2, and IL-16 ELISA for ECP	Positive rates of CCL17/TARC, CCL24/eotaxin-2, and IL-16 were higher in patients with AC, AKC, and VKC compared to controls. Levels of CCL17/TARC, CCL24/eotaxin-2, and IL-16 in patients with AKC and VKC were significantly higher than those in patients with AC. Patients with AKC and VKC showed significant correlation between levels of CCL24/eotaxin-2 and ECP
Inada et al, 201756	This study investigated the histamine H1 and H4 receptors mRNA (H1R and H4R, respectively) expression on the ocular surface of patients with chronic forms of allergic conjunctival diseases in the conjunctiva.	n=7 AKC/VKC stable n=12 AKC/VKC active n=15 control	Real-time RT-PCR H1R, H4R, and eotaxin-1, −2, and −3 Immunohistochemistry for ECP, eosinophil MBP, eotaxin-2, and H4R	The number of H4R-positive patients was higher in the active than the stable stage subgroup and control group, whereas no difference was observed for H1R. H1R levels were higher in the active than in the stable stage subgroup, while those of H4R were higher in the active stage subgroup than in the control group. H1R and H4R levels were correlated with eotaxin-2 level. Immunohistochemical analysis, H4R revealed their expression on in conjunctival smears of patients with AKC/VKC.
Fujishima et al, 201640	Tear periostin in patients with SAC, VKC, and AKC and allergic patients without conjunctivitis and compared it with tear IL-13 and serum periostin. Tear periostin in AKC before and after topical treatment with tacrolimus.	n=26 AKC n=4 VKC n=15 SAC n=14 control	ELISA for periostin and IL-13	IL-13 elevated in tears of AKC, but not VKC or SAC Periostin levels were high in AKC, VKC and SAC Vs control Periostin and IL-13 levels in the tears of all patients with ocular allergic disease and healthy controls showed significant correlation
Dadaci et al, 201570	To evaluate serum MDA, adjusted IMA, and Mg levels in SAC patients	n=35 SAC n=38 control	Mg concentrations with Cobas Integra 400 analyzer MDA with thiobarbituric acid IMA binding of exogenous cobalt to albumin for	Serum MDA and adjusted IMA levels of the subjects with SAC were significantly higher than the control group. There was no significant difference for serum Mg levels between the groups.
Leonardi et al 201544	Measurement of specific IgE in tears using a multiplex allergen microarray	n=10 VKC n=10 control	Multiplex	Normal subjects resulted negative for the presence of specific IgE both in serum and in tears. VKC, six samples were positive to specific IgE in serum and/or tears.
Leonardi et al, 20146	Analyze peptide profiles in human tears using mass spectrometry to elucidate compositional differences between healthy subjects and patients with VKC.	n=10 VKC n=10 control	iTRAQ 4-plex	78 proteins were identified Levels of albumin, transferrin, hemopexin, mammaglobin B, and secretoglobin 1D1 were found significantly higher in VKC compared with normal subjects.
Wakamatsu et al, 201245	Evaluate tear and serum IgE and ECP as severity markers for AKC	n=30 AKC n=10 controls	ELISA for ECP and IgE	Higher total IgE and ECP levels in AKC tears compared with the control group. Tear ECP levels significantly correlated with fluorescein and conjunctival scores (r=0.70 and 0.62, respectively). Tear IgE had no correlation with clinical signs. Seum IgE and ECP levels were elevated in AKC patients but no correlation with clinical signs.
Wakamatsu et al, 2011109	To examine the clinical efficacy and anti-inflammatory effects of tacrolimus eye drops on tear ECP levels in AKC patients	n=9 AKC	ELISA for ECP	Post-treatment tear ECP levels were significantly reduced (195.71±164.46) ng/ml compared to the pre-treatment level (2,680.22±2,342.7 ng/ml. Significant positive correlation between tear ECP with corneal fluorescein staining (r=0.70, p=0.0039).
Wakamatsu et al, 201038	To evaluate the ocular surface lipid oxidative stress status and inflammation in AKC patients and normal subjects	n=14 AKC n=9 controls	Immunohistochemistry for HEL and 4-HNE in brush cytology samples ELISA for HEL and IL-4,IL-5, IL-10, TNF-α, IFN-γ in tears	HEL and 4-HNE significantly elevated in AKC patients. Significantly higher levels of HEL IL-4,IL-5, IL-10, TNF-α, and IFN-γ were detected in tears of AKC compared to controls. Significant linear positive correlation between conjunctival inflammation and epithelial lipid oxidative stress status was observed.
Pong et al, 2010110	Association of hemopexin in tear film in VKC patients	n=29 VKC n=8 control	ELISA	Hemopexin was elevated in the tear film of patients with VKC. The increased hemopexin concentration in VKC tears was significantly associated with disease severity.
Leonardi, 200967	Multiple mediators and growth factors in tears of VKC patients with active disease	n=24 VKC n=12 control	stationary phase antibody arrays.	Interleukin-8 signals were highly detected in all VKC IL-4, IL-5 and IL-10 detected only in VKC bFGF, HB-EGF, VEGF and HGF detected in 41-87% of VKC MMP-1, MMP-2, MMP-3, MMP-9 and MMP-10 were highly present in all VKC samples as compared to control
Shoji et al, 2009111	evaluate the in vivo expression of, eotaxin-1, −2, and −3, patients with VKC and in those of healthy individuals.	n=25 VKC n=11 control	ELISA and RT=PCR for eotaxin-1, −2, and −3	Expression ratio of eotaxin-1 (P < 0.01) and −2 (P < 0.001) significantly higher in VKC compared to control. In the VKC group, the concentration of eotaxin-2 was higher than eotaxin-1. There was a significant correlation between the concentration of eotaxin-2 and ECP in the VKC group
Matsuda, et al, 200919	To study genetic association between the ST2L gene and atopy by looking at expression of IL-33.	n=6 AKC n=5 conjunctivochalasis n=4 superior limbic keratoconjunctivitis n=2 Mooren Ulcer	Immunohistochemistry for IL-33	IL-33 expressed in vascular endothelial cells in the giant papillae and in the control conjunctivae. IL-33 expression was also observed in conjunctival epithelium of the giant papillae but not in the control conjunctivae.
Dogru et al, 200897	Investigate MUC16 and MUC5AC in ocular surface of patients with AKC	n=18 AKC n=14 control	Immunohistochemistry and RT-PCR for MUC16 and MUC5AC in impression cytology samples	Patient eyes showed positive staining for MUC5AC and MUC16. MUC16 mRNA expression was significantly upregulated and MUC5AC mRNA significant downregulation in eyes of AKC compared to control.
Inada et al, 200727	Variations in total secretory IgA and house dust mite specific IgA antibodies in tears of patients with ACD.	n=13 VKC n=11 AKC n=16 PAC n=21 normal	ELISA	Total sIgA values were significantly lower in the VKC group than in the control group
Shoji et al, 2007112	Evaluate the significance of soluble IL-6R in tears as a clinical indicator of disease exacerbation in patients with ACD.	n=13 VKC n=13 AKC n=11 GPC n=10 normal	ELISA	The concentrations of soluble IL-6R in VKC and GPC groups were significantly higher than those in the control group. The concentration of soluble IL-6R and the clinical score of allergic inflammation of the ocular surface were significantly correlated in the VKC group.
Soji et al, 200635	To investigate differences in the cytokine and chemokine profiles of patients with VKC or GPC	n=6 VKC n=5 GPC n=5 normal	40 marker Antibody array	VKC, eotaxin, IL-11, MCP-1, and M-CSF increased four fold the values to control group, VKC, Eotaxin-2, IL-4, IL-6, IL-6sR, IL-7, MIP-1δ,and TIMP-2, increased to eight fold control values GPC, IL-6, M-CSF, and MIG increased to four fold to control group, GPC, eotaxin-2, IL-6sR, IL-11, MIP-1δ,and TIMP-2, increased to eight fold to control values. Increase in IL-6sR compared to controls was statistically significant in both VKC and GPC groups
Dogru et al, 200699	MUC 1, 2 and 4 alterations, tear function and the ocular surface in patients with AKC	n=14 AKC n=11 control	Immunohistochemistry and RT-PCR for MUC 1, 2 and 4 in impression cytology samples.	Specimens from patient eyes showed positive staining for MUC 1, 2 and 4. MUC 1, 2 and 4 mRNA expressions were significantly higher in eyes with significant epithelial disease compared with eyes with insignificant epithelial disease and eyes of control subjects.
Motterle et al, 200673	Expression of neurotransmitters and neurotransmitter receptors in VKC tissues to evaluate whether neurogenic inflammation plays a role in this ocular atopic-related disorder.	n=8 VKC n=4 control	Immunohistochemistry for M1, M2, and M3 muscarinic receptors; β1-adrenergic receptor; vasoactive intestinal peptide; nerve growth factor; and protein gene product 9.5.	M1muscarinic receptor, nerve growth factor, and protein gene product 9.5 expression were decreased compared with control subjects.
Kitaichi, et al. 2006113	To Determine the quantity of MIF in regional ocular fluid of ACDs and AD	n=16 AD n=10 ACD n=15 control	ELISA for MIF	MIF concentration in controls was 0.69+/−0.2 ng/ml Vs 17.87+/−6.3 ng/ml in moderate-to-severe, 0.93+/−0.08 ng/ml in mild atopic dermatitis and 2.76+/−0.86 ng/ml in ACDs
Yamagami, et al. 2005114	To evaluate chemokine receptor genes and their ligand expressions in upper tarsal conjunctival giant papillae of AKC with atopic dermatitis and/or asthma	n=5 AKC n=3 controls	Gene expression for CXCR4 and CCR4, TARC/CCL17, SDF-1/CXCL12, IL-4 and IL-13	CXCR4 and CCR4 are the major chemokine receptor genes expressed in the giant papillae of AKC with atopic dermatitis and/or asthma. Giant papillae with high CCR4 gene expression levels showed high IL-4 and IL-13 expression
Ebihara et al, 200457	Levels of mast cell chymase and tryptase activity in the tears of patients with VKC	n=38 VKC n=14 control	chymase and tryptase enzyme activity	High tryptase and chymase activity in VKC tears compared to control Only chymase activity correlated to clinical signs.
Nivenius et al, 200436	To investigate the possibility that microorganisms may be important in the inflammatory activity in AKC.	n=15 AKC n=12 control	Bead array for IFN-γ, TNF-α, IL-2, IL-4, IL-5 and IL-10	AKC patients showed significantly higher levels of IFN-γ, TNF-α, IL-2, IL-4, IL-5 and IL-10 than controls. An association was found between conjunctival signs and the levels of all cytokines except IL-5.
Leonardi et al, 2004115	Measure Th1-type and Th2-type cytokine and chemokine levels in tears of patients with different types of allergic conjunctivitis	n=18 VKC n=12 SAC n=6 AKC n=3 GPC n=14 control	Multiplexed bead assay IL-;1ß, -2, -4, -5, -6, -8, -10, -12, -13, IFNγ, TNF-α, Eotaxin, RANTES and MCP-1	Controls:low levels of IL-6, IL-8, MCP-1 and RANTES VKC: significant increase over control of IL-1ß, -2, -4, -5, -6, -10, -12, -13, IFNγ, MCP-1 and eotaxin, SAC: significant increase over control of IL-1ß and IL-2, and AKC of IL-1ß, -6, -8 and MCP-1 AKC: significant increase over control of IL-1ß, -6, -8 and MCP-1 GPC: IL-8 was increased over control, VKC and SAC IL-5 was significantly increased in VKC over all the other groups
Leonardi et al, 2003116	Quantify the presence of TNF-a in tear and serum in ACDs	n=12 control n=12 VKC n=6 SAC	ELISA	Serum and tear TNF-α levels in VKC were significantly increased compared to control and significantly correlated with the severity of the disease.
Leonardi et al, 200364	To study levels MMP-1 and −9 and TIMP-1 in tears of patients with VKC, with and without severe corneal damage.	n=16 VKC n=10 control	ELISA MMP-1, MMP-9 and TIMP-1 Activity assay for MMP-1 and MMP-9	Levels of MMP-1 and MMP-9 were significantly increased in patients with VKC compared with control subjects (P < 0.001). MMP-1/TIMP-1 and MMP-9/TIMP-1 molar ratios were significantly increased (P < 0.001) in VKC. MMP-1 and MMP-9 activities were significantly increased in VKC tears compared with control samples (P < 0.005). MMP-9 activity correlated significantly with corneal involvement and giant papillae formation.
Leonardi, et al, 2003117	To measure Eotaxin-1 and Eotaxin-2 in tear and mucus of AKC and VKC patients	n=13 VKC n=3 AKC n=6 control	ELISA Eotaxin-1 and Eotaxin-2 Eosinophil numbers	High levels of eotaxin-1 and eotaxin-2 were found in mucus of VKC patients, only eotaxin-2 was increased significantly in tears of VKC and AKC patients compared with those of normal patients. Tear eotaxin-1 and eotaxin-2 were correlated significantly with the percent of eosinophils. Eotaxin-1 also was correlated significantly with the sum clinical score and corneal involvement in VKC patients
Anderson, et al. 200154	Distribution IL-4, IL-5, IL-6, and IL-13 between mast cell subsets in conjunctival biopsy specimens from nor- mal subjects and those with SAC during and outside of the grass pollen season	n=8 symptomatic, SAC in season n=8 non-sympotmatic, SAC out of season n=22 control	In situ hybridization mRNA IL-4, IL-5, IL-6, and IL-13 Immunohistochemistry IL-4, IL-5, IL-6, and IL-13	> 90% of IL-4+ cells were observed to be mast cells in biopsy of all patient groups. Majority of IL-5+, IL-6+, and IL-13+ cells were mast cells for each group. IL-4 preferentially colocalized to the MCTC with MCTC cells comprising 93.3% of cytokine+ mast cells in symptomatic SAC, 89.2% in asymptomatic SAC, and 77.8% in normal subjects. IL-13 appeared to colocalize preferentially to the MCTC phenotype and IL-5 and IL-6 to the MCT phenotype. 75.8% of mast cells in normal subjects, 78.7% in subjects with symptomatic SAC, and 18.7% in subjects with asymptomatic SAC expressed mRNA for IL-4.
Uchio et al, 200028	Interferon (IFN)-gamma, interleukin (IL)-2, IL-4 and IL-5 levels in tears in ACDs	n=7 VKC n=28 AKC n=14 AC	ELISA	IL-4 level in patients with AKC was significantly higher than those in VKC, AC and controls Tear IL-5 levels in VKC and AKC-proliferative, were higher than those in AC and normal controls.
Leonardi et al, 200053	To measure markers of leukocyte activation in serum and tears of patients with ocular inflammatory	n=17 VKC n=7 AKC n=11 SAC n=7 GPC n=13 BKC n=7 BC n=13 controls	RIA for Neutrophil myeloperoxidase, ECP, eosinophil neurotoxin, and soluble Chemiluminescence assay for IL-2R	In tears of VKC, AKC, SAC, BKC and BC levels of ECP, eosinophil neurotoxin, and IL-2R were significantly increased compared with control subjects with the highest values in VKC. In VKC markers correlateded to the clinical score of the disease. In serum VKC and AKClevels of ECP, eosinophil neurotoxin, and IL-2R were significantly increased compared with control subject but no correlation of marker levels with the severity of ocular symptoms. Neutrophil myeloperoxidase was significantly increased in VKC, AKC, BKC and BC
Fukagawa et al, 1999118	Compare the levels of eotaxin and eosinophils in tears of patients with allergic corneal damage	n=8 controls n=24 AKC (38 eyes in this group 15 eyes with corneal erosion or ulcer (erosion group) 9 eyes with superficial corneal damage (SPK group) 13 eyes with clear corneas (clear group)	ELISA for Eotaxin	Eotaxin in the erosion group (1475.4 ± 870.5 pg/mL was significantly greater than that from the SPK group (163.4 ± 81.7 pg/mL), the clear group (24.3 ± 12.7 pg/mL), and the nonallergic control subjects (21.7 ± 9.7 pg/mL) Erosion group had greater numbers of eosinophils (42.2 ± 52.2 cells/field, n = 9) compared to SPK group (0.3 ± 0.4 cells/field, n = 5), clear group ( (0 ± 0 cells/field, n = 8) and control group (0 ± 0 cells/field, n = 13) The number of eosinophils correlated with eotaxin concentration

AKC (atopic keratoconjunctivitis); ACDs(allergic conjunctival disease); ECP (eosinophil cationic protein); CCL (CC chemokine ligand); Gal-3 (galectin-3); ECP (eosinophil cationic protein); TARC (thymus and activation-regulated chemokine); VKC (vernal keratoconjunctivitis);; H₄R(histamine H₄ receptor); SAC (seasonal allergic conjunctivitis); MCP (mono-cyte chemoattractant protein); M-CSF (macrophage-colony stimulating factor ); MIG (monokine-induced gamma interferon); MDA (serum malondialdehyde), IMA (ischemia modified albumin); HEL (hexanoyl-lysine); 4-HNE (4-hydroxy-2-nonenal); MIF (macrophage migration inhibitory factor); Atopic Dermatitis (AD); GPC (giant papillary conjunctivitis); TNF-α (tumor necrosis factor-alpha); MMP (matrix metalloproteinase); TIMP (tissue inhibitor of MMP); MC_TC(tryptase⁺-chymase⁺ mast cell phenotype); MC_T (tryptase⁺ mast cell phenotype); BKC (blepharokeratoconjunctivitis); BC(bacterial conjunctivitis); RIA (Radioimmunoassay T-helper (TH); SPK (superficial corneal ); MBP (major basic protein), SDF-1/CXCL12 (stromal cell derived factor-1)

Table 2:

Putative “Biomarkers’ for allergic conjunctival diseases

Molecules	References
Th1/Th17 pathway
IFNγ	28,36,115
IL-1β	36,115
IL-6	54,115
IL-8	115
IL-12	115
IL-33	19
TNF-α	36,115,116
Th2 pathway
IL-4	28,36,54,114
IL-5	36
IL-13	40,54,111,114
Eotaxin-1, Eotaxin-2, Eotaxin-3	39,115,117,118
TARC	39,114
Periostin,	40,119
IgE	41,42,45
CCL23	50
Eosinophil activity marker
ECP	32,45,50,53,109,111
Eosinophil neurotoxin	120
Eosinophil granule major basic protein (MBP)	62
Mast cell mediator
Histamine	53,117,121
Tryptase and chymase	53,57,58
LTC4, LTB4	59
H1 and H4 receptors	56
Neutrophil activity marker
Neutrophil elastase, Neutrophil myeloperoxidase	53
PAI-1	7
Proteases and their inhibitors
MMP-1, MMP-9	64
TIMP-2	35,64
PAI-1	7
Oxidative stress
MDA	70
4-HNE, HEL	38,70
Neuromediators
CGRP, VIP, SP	6,73
M1muscarinic receptor, NGF, and protein gene product 9.5	73
Others
MUC 1, MUC2, MUC4, MUC16, MUC5AC	97,99
MIF	113
Gal-3	101
Hemopexin	110

TARC (thymus and activation-regulated chemokine); ECP (eosinophil cationic protein); PAI-1(plasminogen activator inhibitor-1) AKC (atopic keratoconjunctivitis); ECP (eosinophil cationic protein); CCL (CC chemokine ligand); Gal-3 (galectin-3); ECP (eosinophil cationic protein); TARC (thymus and activation-regulated chemokine); H₄R(histamine H₄ receptor); MCP (mono-cyte chemoattractant protein); M-CSF (macrophage-colony stimulating factor ); MIG (monokine-induced gamma interferon); MDA (serum malondialdehyde), IMA (ischemia modified albumin); HEL (hexanoyl-lysine); 4-HNE (4-hydroxy-2-nonenal); MIF (macrophage migration inhibitory factor); Atopic Dermatitis (AD); GPC (giant papillary conjunctivitis); TNF-α (tumor necrosis factor-alpha); MMP (matrix metalloproteinase); TIMP (tissue inhibitor of MMP); MC_TC(tryptase⁺-chymase⁺ mast cell phenotype); MC_T (tryptase⁺ mast cell phenotype); T-helper (TH); MBP (major basic protein), SDF-1/CXCL12 (stromal cell derived factor-1)

(A). Cytokines and Chemokine of the TH1, TH2 and TH17 pathway:

Using ELISA, multiplex bead assay systems, protein arrays and proteomics a number of cytokines and chemokines involved in TH1, TH2 and TH17 immune pathways have been demonstrated in biological samples from ACD patients. Sack et al³⁵, in an early study, looked at the presence of GM-CSF, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8/CXCL8, IL-10, IL-12, IL-13, INF-γ, MCP-1/CCL2 and TNF-α and showed detectable levels of most of these proteins in tears from allergic patients³⁵. In a 40 molecule array study by Soji et al³⁶, expression of eotaxin-1, eotaxin-2, IL-11, monocyte chemoattractant protein (MCP)-1, macrophage-colony stimulating factor (M-CSF),IL-4, IL-6, IL-6sR, IL-7, macrophage inflammatory protein (MIP)-1δ and tissue inhibitor of metalloproteinases (TIMP)-2 were increased more than 4 fold in VKC patient tears as compared to normal. In patients with GPC, IL-6, M-CSF, and monokine-induced gamma interferon⁶, eotaxin-2, IL-6sR, IL-11, MIP-1δ,and TIMP-2, were increased as compared to control values³⁶. Another study found that tear IL-4 levels in AKC patients were significantly higher than those in VKC, AC and controls. Further the IL-4 tear levels differed significantly in AKC patients with proliferative lesions versus VKC patients. Also, tear IL-5 levels in patients with disease associated with proliferative lesions were found to be higher than those in AC and normal controls²⁹. Nivenius et al³⁷ have demonstrated the presence of IFN-γ, TNF-α, IL-2, IL-4, IL-5 and IL-10 in tears from AKC patients. Leonardi et al³⁸, in a comparison of tear cytokine levels between VKC, AKC, PAC and SAC, found significant increase in IL-1β, IL-2, IL-5, IL-6, IL-12, IL-13 and MCP-1/CCL2 in all groups compared to normal. Further, IL-4, IFN-γ and IL-10 were elevated in SAC and VKC, while eotaxin-1/CCL11 and TNF-α were only increased in VKC patient group. Significant differences in expression of IL-5, RANTES/CCL5 and eotaxin-1/CCL11 in VKC patients, compared to SAC patients was also observed³⁸. In another study, IL-4, IL-5, and TNF-α concentrations were significantly higher in the eyes of patients with AKC compared with the eyes of healthy control³⁹. A recent study⁴⁰ has investigated the clinical usefulness of simultaneous assessment of tear levels of CC chemokine ligand 17 (CCL17)/thymus, activation regulated chemokine (TARC), CCL24/eotaxin-2, and interleukin-16 (IL-16) for determining differences in ACDs. They found that tear positive rates of CCL17/TARC, CCL24/eotaxin-2, and IL-16 were higher in patients with AC, AKC, and VKC compared with controls. Tear levels of CCL17/TARC, CCL24/eotaxin-2, and IL-16 in patients with AKC and VKC were significantly higher than those in patients with AC. Moreover, tear levels of IL-16 decreased in patients with AC showing improvement of their clinical score following treatment for 7 days with epinastine ophthalmic solution as compared with levels at baseline. In patients with AKC and VKC, a significant correlation was observed between the tear levels of CCL24/eotaxin-2 and ECP⁴⁰. The conclusion was that simultaneous measurement of the tear levels of CCL17/TARC, CCL24/eotaxin-2, and IL-16 may be a useful test for assessing acute verses chronic allergic inflammation in ACDs⁴⁰. Another recent study showed higher levels of IL-13 in tears of AKC, but not VKC or SAC, while periostin levels were high in AKC, VKC and SAC compared to control⁴¹. Further, periostin and IL-13 levels in tears of ACD patients showed significant correlation with each other⁴¹. Taken together these studies have identified a number of cytokines and chemokines with the potential to serve as biomarkers, especially those that may help distinguish different types of ACDs.

(B). IgE:

IgE is important in the immunopathology of atopic diseases, and is therefore considered as a constitutional marker; both serum and tear IgE levels are routinely measured in the clinic⁴². Using an immunosorbent method, tear IgE concentrations have been shown to be significantly increased in VKC, SAC and PAC compared to normal subjects⁴³. A study has also shown that IgE levels in tear and serum correlate to each other in VKC, AKC and SAC patients⁴⁴. More recently, using a multiplex specific microarray technique for direct measurement of IgE, Leonardi et al have shown the presence of allergen-specific IgE antibodies in tears from active VKC patients⁴⁵. In another study, tear and serum IgE along with eosinophil cationic protein (ECP) were measured to determine their use as severity biomarkers for AKC. Higher levels of total IgE and ECP were detected in AKC tears compared with the control group. While tear ECP levels showed significant correlations with fluorescein staining and oedema scores, tear IgE levels showed no correlation to clinical signs. Serum IgE and ECP levels were elevated in AKC patients but did not show any correlation with clinical signs⁴⁶. While the technology for measuring IgE in an objective and reproducible manner is already available, the studies indicate that its utility as a biomarker for ACDs is inconclusive.

(C). Eosinophil activity markers:

Eosinophils are major constituents of the allergic immune response and therefore proteins associated with eosinophil activity have the potential to be biomarker for ACDs. As such, the presence of ECP and eosinophil neurotoxin (EPX/EDN) in patient biological material is considered a sign of eosinophil activation^47,48. Tear ECP levels have been linked to the severity of eosinophilic inflammation in patients with ACD^33,46,49. It has also been shown that immunosuppressant ophthalmic solutions reduce tear ECP levels⁵⁰. In a study looking at IgE and ECP in tears and serum showed that only tear ECP significantly correlated with patient signs including fluorescein staining, conjunctival injection and oedema scores. This indicated that tear ECP, in contrast to tear and serum IgE and serum ECP, was a good marker to delineate the severity of ocular surface disease in AKC and may therefore be useful in clinical trials and follow-up⁴⁶. In a recent study with chronic ACD patients, tear ECP grade was shown to significantly correlate with clinical severity score. Further clinical severity scores and tear ECP grades in CCL23-positive subgroup among the patients studied was significantly higher than those in CCL23-negative subgroup⁵¹. CCL23 is a member of the CC chemokine family and interacts with the CC chemokine receptor 1 (CCR1)⁵². CCL23 expression has been observed with eosinophils in addition to monocytes and dendritic cells⁵³. In a study with patients with SAC, VKC, AKC and bacterial conjunctivitis, both ECP and EPX/EDN were significantly increased in both serum and tears further underlining the role of eosinophils in ACDs⁵⁴. These studies indicate that ECP may have potential as a biomarker to monitor efficacy of therapeutic agents.

(D). Mast cell derived mediators:

An increase in the number of mast cells in the human conjunctiva is one of the earliest events in the immunopathology of allergy⁵⁵. Consequently, the most common mediator of early phase of hypersensitivity is the histamine released from degranulated mast cells and is therefore a target for the management of a number of allergic diseases⁵⁶. The concentration of histamine produced by basophils in the tears of ACD patients reportedly increases during the late phase of immediate hypersensitivity³³. A number of agonists for H1, one of the receptors for histamine, are used for the management of SAC and PAC³². A recent study⁵⁷ investigating the potential of histamine receptors as biomarkers for ACDs, looked at the expression of H1 and H4 receptors mRNA (H1R and H4R, respectively) on the ocular surface of patients with AKC/VKC. They found that the number of H4R-mRNA positive patients was higher in patients with active stage AKC/VKC than in patients with the stable stage AKC/VKC and control group, whereas no difference was observed for H1R mRNA levels. H1R mRNA levels were also higher in the active stage AKC/VKC than in the stable stage AKC/VKC subgroup, while H4R mRNA was higher in the active stage subgroup than in the control group. H1R and H4R mRNA levels also correlated with eotaxin-2 mRNA levels⁵⁷.

In human there are two mast cell subtypes, mast cell _T (MC_T) and mast cell _TC (MC_TC) that can be differentiated on the basis of the neutral proteases they produced. MC_T subtype express tryptase, while the MC_TC subtype expresses both tryptase and chymase. On the basis of this difference, Ebihara et al⁵⁸ have showed that the levels of tryptase and chymase are increased in VKC patient tears, though only chymase levels show correlation with clinical signs⁵⁸. Another study has also shown increased levels of tryptase in tears of VKC patients and a decrease in levels following treatment with topical 4% disodium cromoglycate and 0.1% fluorometholone eyedrops⁵⁹. Mast cells also produce cysteinyl leukotrienes (LT), LTB₄ and LTC₄ which have been shown in tears of contact lens associated inflammation and ocular prosthesis associated GPC⁶⁰.

(E). Neutrophil activity marker:

Prominent roles for neutrophils in ocular surface diseases like dry eye disease is increasingly being recognized^61,62. Evidence for involvement of neutrophils in ACDs comes from some studies that demonstrated an increase in neutrophil myeloperoxidase in VKC and AKC patient tears as compared to normal⁵⁴. A study by Trocme et al⁶³ shows the presence of extracellular deposition of neutrophil elastase (NE) in conjunctival specimen from patient with VKC and AKC. Notably, when the levels of NE were compared in the same patients with the presence of eosinophil granule major basic protein (MBP), AKC patients had higher levels of NE as compared to MBP while the reverse was true for VKC patient⁶³. It remains to be seen if neutrophil related markers have the potential to serve as biomarkers for ACDs.

(F). Proteases and their inhibitors:

The matrix metalloproteinase (MMP) enzymes are responsible for corneal collagen degradation and inhibited by tissue inhibitors of matrix metalloproteinase (TIMP) enzymes. A study looking at MMP-1, mainly responsible for tissue remodeling, MMP-9, implicated as an inflammatory mediator in ocular surface disease⁶⁴, and TIMP-1 showed that both MMP-1 and MMP-9 are increased in tears of VKC patients. Further, the MMP-1/TIMP-1 and MMP-9/TIMP-1 molar ratios are significantly increased in VKC and MMP-9 activity correlates significantly with some patient signs⁶⁵. A similar observation of higher MMP-1 and MMP-9 in tears of VCK patients was observed in a study by Ghavami et al⁶⁶. They also showed that α−1 Antitrypsin (AAT), a serine protease inhibitor, activity was lower in tears from AKC patients. Another study analyzing tears from VKC found MMP-2 and MMP-9 to be present in all patient samples⁶⁷. It has also been shown that (MMP)-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-10, MMP-13, TIMP-1 and TIMP-2 are present in tears from VKC patients while absent in normal tears⁶⁸. Plasminogen activators, a group of serine proteases, have important roles in facilitating the migration of inflammatory cell, tissue remodeling and cell adhesion among others⁶⁹. A study looking at the presence of urokinase-type (uPA) and tissue type (tPA) plasminogen activators found that these two proteins were highly expressed in conjunctival tissue of VKC patients as compared to normal individuals⁷.

(G). Oxidative stress markers:

Oxidative stress is considered a key event in the pathophysiology of various diseases including allergies⁷⁰. End products generated by modification of lipids, proteins, and DNA by oxidants are used as markers of oxidative stress and include products of lipid peroxidation like hexanoyl-lysine (HEL) 4-hydroxy-2-nonenal (HNE) and malondialdehyde (MDA) and products of protein modification like ischemia modified albumin (IMA). A recent study by Dadaci et al⁷¹ has shown that serum levels of MDA and IMA is higher in SAG patients as compared to control. Similarly, another study has shown that the levels of HEL and 4-HNE are significantly higher in AKC conjunctival cell samples. Further the levels of HEL and 4-HNE correlated positively with levels of IL-5 and TNF-α³⁹. Recent studies with other ocular surface diseases like dry eye disease have also demonstrated elevated levels of HEL, MDA and 4-HNE in patient tears along with positive correlation with a number of patient signs⁷². While more studies are needed with patients from all ACDs groups to determine the validity of oxidative stress markers as biomarkers, on a technical level they may present a challenge due their very short half-lives⁷¹.

(H). Neuro-mediators:

Burning and stinging are key symptoms that characterizes ACDs and are attributed to chronic neuropathic pain. There is increasing evidence that neurogenic mechanisms may have a significant role in allergic inflammation⁷³. A study looking at the presence of M₁, M₂, and M₃ cholinergic muscarinic receptors, β₁-adrenergic receptor (β₁-AR), vasoactive intestinal peptide (VIP), and nerve growth factor (NGF) in the conjunctival cells found that in VKC patients the level of M₁muscarinic receptor and NGF was reduced whereas neurotransmitter receptors and VIP was increased⁷⁴. Sacchetti et al⁷⁵ have shown that following conjunctival allergen provocation test, tear levels of substance P, CGRP and VIP is significantly increased in allergic patients compared to baseline. Another study has shown that levels of Substance P (SP) is high in SAC and VKC compared to normal⁷⁶, while another study has shown increased levels of NGF and SP in plasma from AKC and VKC as compared to matched controls⁷⁷. A better understanding of the ocular surface neuro-immune cross-talk may lead to newer insights into the pathophysiology of chronic neuropathic pain, a hallmark of a number or ocular surface diseases, and perhaps identified biomarkers related to chronic neuropathic pain.

(I). Cell markers.

Evaluation of cell surface markers of inflammation and immune cell phenotypes and their proportions in conjunctival cell preparations are being used to monitor the efficacy of treatment for dry eye disease and other ocular conditions^64,78. The cellular constitution of the tear film has been investigated in patients with ACDs for the presence of neutrophils, eosinophils and lymphocytes⁷⁹. Another study has mapped the cellular profiles of secondary immediate, late and delayed conjunctival responses in the tears induced by a nasal provocation test with allergen⁸⁰. A study has shown that percentages of T cells, activated B cells, and Th1/Th2 cell ratios are higher in tears of patients with AKC than in controls^81,82. The presence of Th2 lymphocytes in tear fluid of patients with VKC have also been demonstrate⁸³.

(f). Others:

Periostin a key mediator in allergic inflammation⁸⁴, is largely produced by epithelial cells, fibroblasts, and endothelial cells in response to IL-4 or IL-13 and has important roles in wound repair^{85,86,87–89}. Periostin overexpression has been shown to plays critical roles in allergic inflammation in atopic dermatitis (AD), and bronchial asthma to name a few^90,91. Periostin is considered a strong potential biomarker in allergic diseases as studies have shown that serum periostin levels are elevated in patients with bronchial asthma and AD^91–93. Measures of serum periostin levels have been used to predict the efficacy of anti–IL-13 antibodies or anti-IgE antibodies therapy^94,95(Corren 2011; Kanemitsu 2013). A recent study by Fujishima et al⁴¹, has shown that tears from patients with ACDs showed significantly higher periostin levels as compared to tears from allergic patients without conjunctivitis. Further, tear periostin level was shown to be associated with serious comorbidities such as corneal damage in AKC. This study also showed that following topical tacrolimus treatment, tear periostin decreased in most patients with AKC and was associated with clinical improvement.

Mucins are glycoproteins present on the ocular surface. Besides functioning as a lubricant with protective functions⁹⁶, there is evidence that mucins also play important roles in immune regulation. Mucins MUC1, MUC4, MUC16, MUC5AC, MUC5B, MUC6, and MUC7 are present in the lacrimal glands⁹⁷. Patients with AKC have been shown to have decreased levels of the goblet cell-specific mucin MUC5AC^98–100 , with concomitant increase in the expression of MUC1, MUC2 and MUC4¹⁰⁰. Anti-inflammatory treatment of VKC patients has been shown to result in a decrease in the levels of MUC5AC levels on the conjunctival surface and in tears^28,101

Recently a role for galectin-3 (Gal-3), a β-galactoside binding protein, as a possible biomarker in ocular allergy has been demonstrated. Conjunctival impression cytology specimens from control and VKC patients showed increased levels of Gal-3. The protein was also increased in murine model of AC. Interestingly the influx of eosinophils, CD4+ expression, levels of eotaxin, IL-4, IL-13 and interferon-γ in the tears of Gal-3^−/− animals with AC was several fold higher than its wild type countertype¹⁰².

Global protein “expression profiling” using proteomics is an approach that is currently being used to study a number of diseases including ocular surface diseses¹⁰³. This approach has the potential to help identify putative ‘markers’, that can be further developed as biomarkers. Using iTRAQ (isobaric tag for relative and absolute quantitation) Leonardi et al⁶ have analyzed quantitative and qualitative proteomic profiles of tears from VKC patients and healthy subjects. They identified 78 proteins differentially expressed in VKC tears. Levels of albumin, transferrin, hemopexin, mammaglobin B, and secretoglobin 1D1 were found significantly higher in VKC compared with normal subjects and of these albumin, transferrin, and hemopexin levels were 10–100 times higher than in normal tears⁶

AKC as a complication of anti-IL-4 and IL-13 therapy for AD

AD, also known as atopic eczema, is the most common inflammatory skin disorder in the world, with a world prevalence of 15–20%¹⁰⁴. AD is a significant cause of ocular morbidity occurring in 20% to 43% of patients^3–5 and AKC is the most severe ocular complication associated with AD. Dupilumab a monoclonal antibody approved in the US and the EU is being currently used extensively for the treatment of atopic dermatitis¹⁰⁵. The mode of action includes binding to the common α chain of the IL-4/IL-13 receptor which results in the inhibition of IL-4 and IL-13 signaling pathways. These signaling pathways have been shown to be involved in B cell differentiation and IgE production, activation of the Th2-dominant immune response and downregulation of filaggrin, a filament-associated protein that binds to keratin fibers in epithelial cells, with subsequent impairment of the skin’s barrier function and the pathophysiology of AD¹⁰⁶. A number of recently published reports have shown a higher incidence of AKC in patients receiving dupilumab. The cause of dupilumab-associated conjunctivitis as well as the relationship between its various pathogenetic mechanisms are currently unclear. One of the hypotheses speculate that inhibition of IL-4 and IL-13 signaling pathways leads to a deviation in the OX40L/OX40 signaling pathway which has shown has to be important in the pathogenesis of atopic keratoconjunctivitis¹⁰⁷ and another proposes a transient dupilumab-induced increase in eosinophils, key cells in the pathophysiology of AKC¹⁰⁸. Studies investigating AKC on a background of IL-4/IL-13 suppression for AD provides a unique opportunity for novel insights into the pathophysiology of AKC in specific and ACDs in general.

Conclusion

Studies to date have added to the extensive database on the biological processes associated with ACDs and provided critical insights into the pathophysiology of ACDs. Based on these studies a number of strong potential ‘biomarker’ candidates have emerged. However, a number of important aspects have to be addressed before one or combinations thereof of these potential ‘biomarker(s)’ meets the definition of ‘a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes or biological responses to a therapeutic intervention’. These includes studies with larger patient cohorts, demonstration of reproducibility of methodology and data obtained, establishing the use of the biomarker, i.e., for diagnosis, establishing severity, assessing change, clinical trial surrogate endpoint and ease of use in a clinical setting. It is hoped that studies with larger patient cohorts that also combine the ‘omics’ approach for wider information will continue to add to our understanding of ACDs and address the important criteria needed to validate ‘biomarker’ candidates for diagnosis, management and assessment of therapeutic effect.