Cornea - ARVO

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaPurpose: Keratoconus (KC) is a corneal disease, which ischaracterized by corneal thinning leading to loss of visual acuitythrough ectasia and irregular astigmatism. Clinically, the symptomsof KC can be highly variable and in part, depend on the stage of theprogression of the disorder as well as associated eye-rubbing, itching,etc. There is currently no effective management for KC, nor anyadequate biomarkers to predict the outcome of clinical severity of thedisease. Furthermore, there is considerable confusion in the fieldregarding the pathophysiology of the disease and key involvement ofinflammation. To that end, this study was designed to address thesequestions.Methods: We correlated the clinical features associated with KC in acohort of 54 patients with tear proteomic profiles. This noninterventionalstudy was approved by the Narayana Nethralaya IRB.Clinical grades and associated symptoms were graded as per theAmsler-Krumeich classification (Grade I: 32 patients, Grade II: 14patients, Grade III: 8 patients). Quantitative Proteomic analysis wascarried out using iTRAQ with nanoLC-MS/MS on 25μg total tearprotein from each patient and pooled healthy control subjects (n = 12)for comparison.Results: We observed that eye-rubbing correlated with increasingclinical grades and morbidity of KC. In total, over 1000 tear proteins(< 1% false discovery rate) were identified from the whole study.Quantitative proteomic results from a group of up-regulated anddown-regulated proteins (ratio for KC/control > 1.5, or < 0.67)revealed a positive correlation between several tear proteins (LCN1,PLA2G2A, SCGB2A1, MSLN and CRYAB) and KC clinical grades(Grade I, II and III). A negative correlation between a group of tearproteins (ALB, IGHG2 & G3, A2M, complement factors C3, C4A,C6 & H, ORM1, KNG1, PRDX1, SERPIN-F1, GSN) and KC clinicalgrades was also observed.Conclusions: These proteins could be used alone or in combinationas biomarkers for predicting the progression or severity ofkeratoconus. These proteins also shed light on the underlyingderegulation of important inflammatory and immunomodulatorypathways that may be key drivers of KC pathophysiology.Commercial Relationships: Arkasubhra Ghosh, None; RohitShetty, None; Lei Zhou, Singapore Eye Research Institute (P);Sharon D'Souza, None; Roger W. Beuerman, Allergan (F), SERI(P), Santen (R)Clinical Trial: J0002GQQProgram Number: 929 Poster Board Number: B0234Presentation Time: 1:00 PM - 2:45 PMSeveral Tear Protein Levels Of Breast Cancer Patients Differfrom Healthy Participants: A Microarray StudyKsenia Keller 1, 2 , Julia Pieter 2 , Daniel Boehm 2 , Norbert Pfeiffer 3 ,Franz H. Grus 1 . 1 Experimental Ophthalmology, University MedicalCenter, Mainz, Germany; 2 Gynecology and Obstetrics, UniversityMedical Center, Mainz, Germany; 3 Ophthalmology, UniversityMedical Center, Mainz, Germany.Purpose: Currently no molecular biomarkers for early detection ofbreast cancer (BC) are available. The explorations of cancerproteome revealed several new findings according significant levelchanges of proteins. Even in the distant body fluids like tears wereported over 20 de- or increased proteins in pooled BC and healthy(CTRL) samples in our previous study. Here we report our additionalstudy regarding individual tear protein profiling using high sensitiveantibody-microarray plattformMethods: Tear fluid was obtained from 38 women, whereas 19 ofthem were diagnosed with primary non-metastasized BC, with thehelp of Schirmer test. Tear proteins were eluted overnight with 0.1%n-dodecyl-beta-D-maltoside. Proteins were precipitated and theirconcentration was determined. 5 µg of labeled proteins from eachparticipant were incubated on microarrays with antibodies againstcomplement proteins, other immune components and high abundanttear proteins. After signal visualization a semi-quantitativecomparison of protein levels was performedResults: We detected several significantly different distributedproteins in BC and CTRL groups. For example, the interleukins IL2and IL1beta were decreased in BC samples (p=0.025 and p=0.017,respectively). Furthermore we used the artificial neuronal networks totest the discrimination ability of the putative biomarkers. Thus thebest combination of biomarkers revealed the sensitivity of 89% andspecificity of 100%. An area under the curve of 0.91 was achievedConclusions: This pilot study provides more findings to our previoustear protein profiling investigations. Obviously also in other bodyfluids besides serum and tumor microenvironment several proteinderegulations occur leading perhaps to changed signal cascades andaberrant reactions. This study also shows the immediate involvementof immune system, whereas its component levels are changed incomparison to CTRL subjects. Further explorations are ongoing forthe verifying the results in a larger study population for establishmentof putative BC biomarkersCommercial Relationships: Ksenia Keller, None; Julia Pieter,None; Daniel Boehm, None; Norbert Pfeiffer, Sensimed AG (F),Sensimed AG (R), MSD (F), MSD (R), Alcon (F), Allergan (F),Novartis (F), Novartis (R), Bayer (F), Heidelberg Engineering (F),Bausch&Lomb (F), Boehringer-Ingelheim (F), Carl Zeiss Meditech(F), Chibret (F), Nidek (F), Pfizer (F), Santen (F), Santen (R),Topcon (F), Ivantis Inc (F), Ivantis Inc (R); Franz H. Grus, NoneSupport: partly founded by MAIFORProgram Number: 930 Poster Board Number: B0235Presentation Time: 1:00 PM - 2:45 PMMethod development for analysis of tear proteins using selectedreaction monitoringSimin Masoudi 1, 2 , Ling Zhong 3 , Mark Raftery 3 , Mark D. Willcox 2 .1 Brien Holden Vision Institute, Sydney, NSW, Australia; 2 School ofOptometry and Vision Science, University of New South Wales,Sydney, NSW, Australia; 3 Bioanalytical Mass Spectrometry Facility,University of New South Wales, Sydney, NSW, Australia.Purpose: Previous studies of dry eye and contact lens (CL) relateddry eye have shown qualitative differences in lactoferrin, lysozyme,lipocalin 1, prolactin-induced protein and proline rich protein 4. Theaim of this study was to develop a method for simultaneous detectionand accurate quantification of these five proteins in human tearsusing selected reaction monitoring (SRM) mass spectrometry.Methods: Basal tears were collected from normal subjects (male=1,female=6, CL=3, No CL=4).Tears were processed as describedbefore with modifications. SRM transitions from two peptidesrepresenting each protein were selected using Skyline software thenanalyzed using nano-flow liquid chromatography mass spectrometry.Calibration standards were generated using unlabelled synthetizedpeptides diluted to a range of 1 to 1000fmol/µL in the presence of afixed amount of stable-isotopically labelled peptides (50fmol/µL).The ratios of the peak areas of the unlabelled/labelled peptides wereplotted against the concentrations of the corresponding unlabelledpeptides. The concentration of endogenous proteins (µg/µL) in tearsamples was deduced using the peak area ratio of the endogenouspeptides to labelled peptides.Results: The limits of quantification for the selected peptides werebelow 50pg/μl. The recovery of peptides from spiked digested tearswas ≥56% and the coefficient of variation values were ≤16% whichshow good precision of the method. For lactoferrin(1.20±0.77μg/μL), lysozyme (2.11±1.50μg/μL) and lipocalin-1©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.