Ocular Surface and

A. J. Bron Nuffield Laboratory of Ophthalmology Oxford

Image -freedoncurrent.com EMA 27/28 th October Biomarkers in Inflammation

• Scope • Technologies • Definition of a biomarker • Monitoring - Candidates • Applications • Diagnosis – • Risk factors v Screening Bioinformatics • Ocular Phenotypes • Duration of trials • Measuring Symptoms • Conclusions • Signs v Symptoms • Sampling variables Scope

•Prenatal screening: • Huntingdon mutation in •Neonatal screening HD – endocrine and metabolic • Serum anti-citrullinated disorders, lysosomal storage peptide plus RhF in dis. •Adult diagnosis diagnosis (PPV 100%) •Alzheimer’s diagnosis: • Prediction of morbidity/ –CSF: A β and τ; FDG-PET mortality in end stage scan renal failure. •HER2 –efficacy of HER2 blockade in treatment of metastatic breast cancer Definition and Applications

• A disease- • Predicting Risk associated • Screening • Diagnosis parameter • Scaling severity • Discriminates • Monitoring progress affected from • Predicting response to therapy unaffected • Determining prognosis • Understanding disease mechanism Prediction of dry eye in at -risk groups? • Contact lens wear • Isotretinoin therapy -MGD • LASIK -Refractive laser surgery – dry eye or LINE • Chronic topical preservatives - in glaucoma therapy • Bone marrow transplantation – G v H disease • Connective tissue disease - 2°rheumatoid Sjögren • Postmenopausal estrogen therapy • Meds: antihistamines • Androgen deficiency or receptor blockade Is a strong risk factor of use in screening:? • The relative odds for the association of

cholesterol (RO1-5) with Ischaemic Heart Disease ≅ 2.7

• This gives a DR5 ≅ 15% which is poor for a screening test

DR = Detection Rate at a False Positive rate of 5% 5 Wald et al. BMJ 1999; 319 Odds ratios and Detection Rates • Emerging Risk Factors Collaboration: CRP and CHD Kaptoge et al 2010 – Odds ratio 3

• Rotterdam Coronary Calcification Study: CC and CHD Vliegenthart et al. 2005 – Relative risk 8.3

• Atherosclerosis Risk in the Community: HbA1C- DM and CHD Selvin et al. 2010 – Odds = 103.5 [for Diabetes]

See Wald and Morris 2011 Arch Intern Med 2011; 171 : www.wolfson.gmul.ac.uk/rsc/ Odds ratios and Detection Rates • Emerging Risk Factors Collaboration: CRP and CHD Kaptoge et al 2010 – Odds ratio 3

– DR5 = 9% • Rotterdam Coronary Calcification Study: CC and CHD Vliegenthart et al. 2005 – Relative risk 8.3

• Atherosclerosis Risk in the Community: HbA1C- DM and CHD Selvin et al. 2010 – Odds = 103.5 [for Diabetes] Wald and Morris 2011: www.wolfson.gmul.ac.uk/rsc/ Odds ratios and Detection Rates • Emerging Risk Factors Collaboration: CRP and CHD Kaptoge et al 2010 – Odds ratio 3

– DR5 = 9% • Rotterdam Coronary Calcification Study: CC and CHD Vliegenthart et al. 2005 – Relative risk 8.3

– DR5 = 22% • Atherosclerosis Risk in the Community: HbA1C- DM and CHD Selvin et al. 2010 – Odds = 103.5 [for Diabetes] Wald and Morris 2011: www.wolfson.gmul.ac.uk/rsc/ Odds ratios and Detection Rates • Emerging Risk Factors Collaboration: CRP and CHD Kaptoge et al 2010 – Odds ratio 3

– DR5 = 9% • Rotterdam Coronary Calcification Study: CC and CHD Vliegenthart et al. 2005 – Relative risk 8.3

– DR5 = 22% • Atherosclerosis Risk in the Community: HbA1C- DM and CHD Selvin et al. 2010 – Odds = 103.5 [for Diabetes] Wald and Morris 2011: www.wolfson.gmul.ac.uk/rsc/ – DR5 = 32% Screening for Downs and Neural Tube Defect • 2-step integrated test US - Nuchal translucency in Downs for Downs • 1st Trimester –nuchal translucency and serum - associated plasma protein A • 2nd Trimester - AFP, hCG, unconjugated Serum AFP raised in NTD estriol, and Inhibin-A Nearly all NTD can be identified by AFP screening. • Risk result in 2 nd tr. DR5 = 91% spina bifida • DR = 90% Valuable2 diagnostic tests may take time to Wald 2010 Global Phenotypes DRY EYE features Symptoms +

Hyperosmolarity +

Tear Instability +

Surface stain +

Tear allergy markers negative Global Phenotypes DRY EYE features Symptoms + > 20 OSDI Hyperosmolarity + ≥ 316 mOsm/l

Tear Instability + BUT ≤ 10 s

Surface stain + ≥ 3 or 4 VB

Tear allergy markers negative Phenotypes DRY EYE - aqueous deficient Symptoms + > 20 OSDI Hyperosmolarity + ≥ 316 mOsm/l +ve –ve Schirmer Tear Instability + MGD ≤ 5 mm BUT ≤ 10 s negative MGD Meniscus radius surrogates Meniscus height Surface stain + Tear clearance VB Tear EGF ≥ 3 or 4 Tear Lysozyme Tear Lactoferrin Tear allergy markers negative Phenotypes DRY EYE - aqueous deficient Symptoms + > 20 OSDI Hyperosmolarity + ≥ 316 mOsm/l +ve –ve Schirmer Tear Instability + MGD ≤ 5 mm BUT ≤ 10 s negative MGD Meniscus radius surrogates Meniscus height Surface stain + Tear clearance VB Tear EGF ≥ 3 or 4 Tear Lysozyme Tear Lactoferrin Tear allergy markers negative Phenotypes DRY EYE Evaporative Symptoms + > 20 OSDI Hyperosmolarity + ≥ 316 mOsm/l

Schirmer Tear Instability + MGD + >5 mm MGD signs + BUT ≤ 10 s ↑evaporation TFLL changes - negative LG Meibum change surrogates Surface stain + tear Calgranulin ≥ 3 or 4 VB

Tear allergy markers negative Phenotypes DYSFUNCTIONAL TEAR SYNDROME Symptoms + > 20 OSDI Hyperosmolarity + ≥ 316 mOsm/l

Schirmer Tear Instability + MGD + Lack of expressable < 10 mm BUT ≤ 7 s meibum ≥ 75% of glands

Surface stain + 2 or more of: VB Acinar atrophy ≥ 3 or 4 Orifice metaplasia Vascular dilatation at posterior lid margin Tear allergy markers negative Endpoints – Signs versus Symptoms symptomssymptoms inin drydry eyeeye  Soreness, irritation  Frequency  Gritty, scratchy  Timing  Burning, stinging  Intensity  Itching  Dryness  Provocations:  Tired eyes.  Low humidity-AC  Airflow – windy day  Fumes - smoke  Light Sensitivity,  Visual Change eg D E Q– Begley et al 2002 Symptom Name # of Author questio Measurement Womens ns3 Schaumberg et al. 2003 • In dry eye, whose major Health Sjögren 3 Vitali et al. 2002 feature is symptoms, Consensus there is no surrogate for Schein 6 Schein et al. 1997 symptom measurement McMonnies 12 McMonnies and Ho 1986 • Validated OSDI 12 Schiffman et al. 2000 Questionnaires are SPEED 12 Korb et al. 2005 available CANDEES 13 Doughty et al. 1997 • Biomarkers whose DEQ 21 Begley et al. 2002 levels correlate with NEI-VFQ 25 symptom severity are of OCULAR 31 Johnson Murphy COMFORT 2007 interest because they INDEX may be closer to IDEEL 57 Rajagopalan et al. symptom mechanisms 2005 Symptom / Sign correlation is often poor • Goren 1988 • Enriquez de Salamanca • Begley 2003 2010 • Nichols 2004 • No correlation with global • Saleh 2006 scores: • Moore 2009 • Some scattered corrlns with individual CKs. • Fuentes-Paez 2011 Symptom sources in dry eye

 Hyperosmolarity  Diffuse: meniscus sample  Focal: tear film break up [Ocular Protection Index - BUT/Blink interval]. 1  Reduced lubrication  frictional drag: loss of glycocalyx and goblet cell mucin  lid wiper epitheliopathy. 2 [Shearing between lids and globe during blinks and eye movements]  Conjunctivochalasis  Inflammatory mediators [Prostanoids, cytokines, neurokinins, neuromediators]  Ocular surface damage [Alterred nerve excitability 3; neuropathic firing 4] 1. Ousler et al. 2008 2. Korb et al 2005 3. dePaiva and Pflugfelder 2004 4. Belmonte, Gallar 2011 . Symptom sources -- areare dependentdependent onon --

Corneal sensory fibres – Polymodal nociceptors – Cold fibres 1 Muller et al. 2005 • Physiological De Paiva Pflugfeldedr 2004 – Surface stress - increased stimuli – increased excitability • Neuropathic firing – cold fibres 1 Lid margin sensory fibres?

1. Belmonte Gallar IOVS 2011, Vol. 52, 3888 the source of symptoms in dry eye

Innervation of the Eye Sensitivity varies over the lids • ManyLack of sources a powerful of symptoms and ocular surface

whoseassociation relative between contribution a conjunctiva maybiomarker change and with dry stage eye of disease.symptoms at diagnosis

• Currentshould not symptoms discourage may its cornea 64% usereflect to trackthe cumulative 6.0the efficacy effect of a of drug, several causes. Bulbar conjunctiva • particularlyStudies needed where to itidentify reflects alid/MGD causal specifichypothesis symptoms or could tarsus

provide proof of principle of Lid margin

drug action Lid margin mucosa Sensitivity of Lid margin skin Lid margin mucosa Lawrenson and Ruskell 1993; as high as Cornea McGowan et al 1994 Tissue sampling - variables affecting measurement TearEpithelial samples Cell Samples • CapillariesImpression vcytology absorbent materials; eye wash • Available– Instant, regionalvolume sample of surface cells • Brush– ADDE cytology low; EDE normal? • Reflex– Global tearing sample and sample dilution. • Analysis– ADDE – falls with severity; EDE rises? • Value– Immunocytochemistry of tiny, nL samples - repeatability – InstantFlow cytometry analysis (osmolarity); or multistep • Ocular– HLA-DR; surface mRNA; permeability-molecular cytokines; transmembrane size mucins of • Standardisationbiomarker is the key – optimize techniques to– conjunctivaenhance repeatability/ cornea; vascular/epithelial. . • Biomarker ratios in single samples Molecular Biomarker Technologies • Electrophoresis: 1D; 2D gels • Bioinformatics – • ELISA sandwich protein networks. • Protein arrays (beads, blots) • Western blot • LC-MS • SELDI/TOF • MALDI/TOF • LC MALDI

• LC-MS/MS Waters • iTRAQ CandidateMultiplex Bead Assay Tear / Microwell Proteins and membrane Arrays - R Sack • CYTOKINES • OTHER molecules • lL-1α; lL-1β; 2; 4; • Soluble receptors: 5;6;8;10; IL-1RI, II; IL-2R, γ; IL- 12P70;13;15;17;23 4R; IL-6R; IL-6R; IL- • INFγ; TNFα; TNFβ 13Ra1; TNF-R1; • CHEMOKINES TNF-RII; • Eotaxin; GROα; I-309; • Sgp 130; gp340 IL-8; IP10; MCP-1,2; • α2-M RANTES; TARC • ADHESION molecules • ICAM-1, 3; VCAM-1; • E-,L -,P- selectin, Candidates: Chemokines in Dry Eye: Th-1 -dependent inflammation Yoon IOVS 51 643 2010

Chemokine type CXC [ ααα] CC [ βββ] C [ γγγ] CXXXC [ δδδ] Control ELR ELR + - IFN γγγ

CXCL9 [MIG] CXCL10 [IP-10] CXCL11 [I-TAC] Non-Sjogren DE Receptors CXCR 1 CXCR 3 & 5 CCR 3 & 4 and 2 T-cells * Recruits PMNS NK cells Sjogren DE

Th-1 related Th-2 related inflammation inflammation Candidates: Chemokines in Dry Eye Th-1 -dependent inflammation Yoon IOVS 51 643 2010

• Capillary tears: ELISA; • CXCR3 + CD4 + conj. cells – CIC flow cytometry. main effectors of lac. and • Increase in: conj. epithelial damage? – IFNγ -inducible • CXCL 11 levels correlated ELR- CXC with chemokines in DE – low basal Schirmer, tears. CXCL 9, 10 – low tear clearance, esp 11 , and – kerato-epitheliopathy, + – CXCR3 Th 1 type – reduced goblet cell cells in conj. density. epithelium. Candidates: Cytokine profiles in Dysfunctional TS Lam et al. 2008

• Subjects: 30 DTS; 14 • IL-1Theseα,β cytokines, IL-6, 8 & control (CXCL8)MIP-1α correlated IL-10, • 2-eye, pooled 0.5 µl with12(p70), DEWS 13 severity tear capillary samples • IFNgrade:α;TNFα • Luminex Bead array • IL-6Macrophagecorrelated inflam with proteinseverity ( MIPof symptoms-1α) andCCL3 signs • EGFRANTES levels CCL5 correlated • EGFwith the Schirmer value and inversely with corneal staining. Candidates: Cytokine profiles in Dysfunctional TS Lam et al. 2008

• Subjects: 30 DTS; 14 • IFNγ / IL-13 ratio ↑ control in DTS • 2-eye, pooled 0.5 µl • IFNγ a marker for Th-1 tear capillary samples inflammation and IL- • Luminex Bead array 13 for Th-2 inflammation • The ratio correlates with goblet cell loss and metaplasia in DE model Candidates: MMP9 in Dysfunctional TS Chotikavanich et al. 2009

• Subjects: 19 DTS; 16 control (+subset) • 2-eye, pooled 0.5 µl tear capillary samples • Tear immunoassay, CIC RNA real-time PCR Candidates: MMP9 in Dysfunctional TS Chotikavanich et al. 2009

• Tear MMP9 activity ↑ in IncreasedAlso correlates RNA epithelial with: transcripts in DTS DTS patients; correlated • Surface stain; confocal with: epithel. score; surface – Increases in -IL-1β; IL- irregularity; low contrast 6 ; TNFα AND TGFβ1 sensitivity. CIC epithelial • No corrln with BUT. transcripts. but -MMP9 also increased in – Clinical severity patients with MGD and with controls = 8.4 pg/ml SS [Solomon 2001 IOVS 42 2283] . and proMMP9 is DTS grade 4 = 381.2 increased in rosacea [Afonso pg/ml P<0.001] 999 40 2506; Sobrin IOVS 2000, 41 1703] Candidates: tear and membrane bound MUC1 Caffery 2010 • The trans membrane Argueso Gipson 2001 mucin MUC 1 is a key MUC 1 component of the ocular surface glycocalyx. • Cleavage of the exodomain releases soluble MUC1 into the

tears. Ocular surface mucins are: MUC1, MUC2, MUC4 , MUC5AC, MUC7, MUC13, MUC15, MUC16, and MUC17. Goblet cell Candidates: tear and membrane bound MUC1 Caffery 2010 • Subjects: 25 primary SSDE; 25 NSDE; 26 controls • Eye wash and pooled CIC samples

• Tear MUC1 and MUC1 expression highest in SSDE. Tear MUC1 also higher in NSDE Candidates: tear and membrane bound MUC1 Corrales 2011

• Subjects: 38 • Expression of MUC 1, 2, NSDE; 43 4 and 5AC lower in controls. NSDE • Individual CIC • Using MUC1 expression samples in dry eye diagnosis:

DR12.5 = 83.3 % • Validated in additional control and DE groups. SELDI -TOF-MS Protein Chip Array in dry eye diagnosis • Focus on Mass < 14 KDa. • Multivariate discriminant analysis used to identify 50 peaks differing between ADDE and normals

Grus 2005 SELDI -TOF-MS Protein Chip Array in dry eye diagnosis • Focus on Mass < 14 KDa. • Multivariate discriminant analysis used to identify 50 peaks differing between ADDE and normals • Further analysis revealed a cluster of 7 polypeptides •GrusDry 2005 eye detection rate iTRAQ technology with 2D-nanoLC- nano-ESI-MS/MS Zhou Proteome Res 2009

• Subjects: 56 DE: Symptoms+; Sch ≤10 mm; FBUT ≤ 10s; Cr Stain >2 Oxf • 40 control • 10 mm Schirmer strip sample

• 93 tear proteins identified, 10 differentially expressed iTRAQ technology with 2D-nanoLC- nano-ESI-MS/MS Zhou Proteome Res 2009 • 6 up-regulated proteins , • Diagnosis with a 4 • α-enolase, protein biomarker • S100 A4 and panel: • α-1-acid glycoprotein 1, DR10 : 91% • S100 A8 (calgranulin A), • S100 A9 (calgranulin B), • S100 A11 (calgizzarin) • 3 proteins: • 4 down-regulated • α-1-acid glycoprotein 1, • lactoferrin and lysozyme. • S100 A8 (calgranulin • prolactin-inducible protein A), (PIP), • S100 A9 (calgranulin • lipocalin-1 B), • Correlated with severity iTRAQ technology Tong et al. MGD and Dry Eye 2011

• Subjects: 24 DE: • Calgranulin A and B Symptoms+; Sch ≤10 ratios correlated with: mm; FBUT ≤ 10s; Cr • MGD severity and Stain >2 Oxf; • Symptoms : Redness; • MGD severity scale 0-3 transient blurring • Lipocalin-1 was • 18 control associated with heaviness • Schirmer strip sample of the eyelids and tearing • “MGD may independently contribute to the symptoms of dry eye patients”. Cytokines - Antibody Microarray Aqueous- and lipid-deficient Dry Eye

• Subjects: ↑ IL-1,-6,-8 TNF α, IFN γ, LCN-1, Cystatin SN, α1-AT • 35 DRYaq; In aqueous deficient not lipid deficient dry eyes • 36 DRYLip; • 34 mixed • 38 Controls. • Eluted Schirmer strips • Antibody microarray Boehm IOVS 2011 Recommendations • Establish : • Apply to broad – Rigorous criteria for population samples each phenotype with dry eye and other – Validated ocular surface Questionnaires disease. – Measures of severity • Establish cut offs. • Optimize tissue • Validate key sampling biomarkers or panels – nano volumes; cell snapshots • Refine diagnostic and severity criteria • Select biomarker technology with low variance in field conditions. Thank You for your Attention