CLINICAL SCIENCES Melanoma-Associated Retinopathy A Paraneoplastic Autoimmune Complication Ying Lu, MD, PhD; Lin Jia, MS; Shirley He, MD, MS; Mary C. Hurley, MS; Monique J. Leys, MD; Thiran Jayasundera, MD; John R. Heckenlively, MD Objectives: To study 11 patients with melanoma- patients (9 of 11) had a strong family history of autoim- associated retinopathy (MAR) to clarify the reliability of mune disorders. Any type of melanoma (cutaneous, cho- various methods of diagnostic testing, to determine the roidal, ciliary body, or choroidal nevi) may be associ- underlying antigenic retinal proteins, and to study the ated with this paraneoplastic autoimmune reactivity. MAR clinical histories and types of associated melanomas. may precede or follow the diagnosis of melanoma. Pa- tients with MAR have the same antigenic retinal pro- Methods: Clinical data were obtained from patients with teins that have been associated with cancer-associated reti- melanoma who developed marked visual problems. Test- nopathy. In addition, 2 new antigenic retinal proteins, ing included electroretinography, kinetic visual fields, aldolase A and aldolase C, were found. comparative studies of Western blots, and indirect im- munohistologic examination to detect antiretinal anti- Conclusions: There was a high prevalence of positive bodies, as well as proteomic studies to identify underly- family histories of autoimmune disease in patients with ing antigenic retinal proteins. MAR. To confirm the disorder, multiple clinical and se- rum diagnostic techniques (Western blot or indirect im- Results: Patients with MAR typically have rapid onset of photopsias, scotomata, and loss of central or paracen- munohistologic examination) are needed. Two newly ob- tral vision. Ophthalmoscopy seldom shows significant served antigenic retinal proteins, aldolase A and aldolase changes early, but electroretinograms are abnormal. Re- C, are associated with MAR. sults of Western blots and immunohistologic examina- tion can show antiretinal antibodies but not always. Most Arch Ophthalmol. 2009;127(12):1572-1580 UTOIMMUNE RETINOPATHY with carcinomas,1 melanomas,8 tetrato- (AIR) was first recognized mas,2 and even lymphomas.9 Most cases of as a paraneoplastic disor- AIR occur in patients without a history of der as early as 1976 in pa- tumors, but occasionally a history of head tients with carcinoma,1 and trauma or preceding intraocular inflamma- Asubsequent studies in the literature showed tory disease may be found.10-12 Investiga- that AIR could occur with benign2 and ma- tions of cancer-associated retinopathy lignant3 tumors. Melanoma-associated reti- (CAR) have found that the tumors in these nopathy (MAR) was initially reported by patients aberrantly express proteins nor- Gass4 in an atypical case with vitellirup- mally exclusive to retinal tissue, leading to tivelike yellow retinal lesions, but most MAR the production of antibodies directed cases have diffuse retinal atrophy without against these retinal antigens.13 There are pigment deposits. A broader view of 51 cases no reports in which primary melanomas 5 Author Affiliations: of MAR was published by Keltner et al sum- have been examined to investigate the pres- Department of Ophthalmology marizing many of the features and some of ence of retinal proteins, although mela- and Visual Sciences, Kellogg the controversies. Except for 2 case re- noma cell cultures have demonstrated their Eye Center (Drs Lu, He, ports of choroidal melanoma,6,7 only MAR presence.14 The antiretinal antibodies in pa- Jayasundera, and Heckenlively associated with cutaneous melanomas has tients with MAR are associated with pro- and Ms Jia), and Michigan been reported.5 gressive panretinal degeneration that fre- Proteome Consortium Paraneoplastic development of antireti- quently results in legal blindness.15 Patients’ (Ms Hurley), University of Michigan, Ann Arbor; and nal antibodies with resulting retinal dis- early symptoms typically manifest as Department of Ophthalmology, ease is an intriguing pathologic process and sparkles and shimmers (photopsias) and 16 University of West Virginia, remains poorly understood. There have blind spots in their vision. On clinical ex- Morgantown (Dr Leys). been reports of retinopathies associated amination, early stages of retinopathy may (REPRINTED) ARCH OPHTHALMOL / VOL 127 (NO. 12), DEC 2009 WWW.ARCHOPHTHALMOL.COM 1572 ©2009 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/29/2021 be difficult to see clinically, but an electroretinogram (ERG) mobilized pH gradient buffer). The solubilized protein was loaded (a standardized evoked response that measures photore- overnight onto 11-cm (pH 3-10) immobilized pH gradient strips ceptor and inner retinal responses) will clearly show reti- using active rehydration at 50 V. Isoelectrofocusing was per- nal dysfunction and aids in diagnosis of this condition with formed at 40 000 V/h using isoelectric focusing Cell (BioRad In- otherwise minimal retinal changes.17 dustries, Hercules, California). The focused IPG strips were equili- brated in buffers containing sodium dodecyl sulfate (SDS)-Tris Conventional confirmatory evidence for MAR has in- (2-carboxyethyl)phosphine hydrochloride and SDS- cluded examination of serum for antiretinal antibodies iodoacetamide. The second-dimension gels were run on 4% to by Western blot or measurement of antibody staining of 12% acrylamide Bis-Tris gels (BioRad Industries). After electro- donor retina, particularly bipolar cells, by indirect im- phoresis, duplicate gels were inmmunoblotted or stained with munohistologic studies.18 However, there has been con- SYPRO Ruby (Molecular Probes, Eugene, Oregon). troversy about the best diagnostic tests, with data sug- Separated proteins were transferred onto nitrocellulose mem- gesting that Western blots may be unreliable for detecting branes and incubated with serum from patients having MAR antiretinal antibodies in patients with MAR.8 Milam et and with horseradish peroxidase–conjugated mouse antihu- al8 recommended indirect immunohistologic investiga- man immunoglobulin as a secondary antibody. Immunoreac- tions for retinal inner nuclear layer bipolar cell staining tive spots on each membrane were compared visually and by using a computerized system. Each of the spots was matched as a reliable technique for confirming MAR. To date, no to an equivalent spot on staining gels. studies have been performed in patients with MAR com- paring Western blot with indirect immunohistologic de- IN GEL ENZYME DIGESTION tection of antiretinal antibodies. In addition, the spe- AND MASS SPECTROMETRY cific antibodies (and their antigenic retinal proteins) involved in MAR have not been systemically examined. Spots from 2-dimensional electrophoresis–stained gels were ex- We investigated 11 patients with MAR who were ini- cised and in gel digested (Trypsin Gold; Promega, Madison, Wis- tially seen with visual loss and photopsias and who un- consin). Peptides were extracted from the gel plugs in 30 µL derwent clinical examinations, electrophysiological and of 2% acetonitrile, 1% formic acid. Five microliters of alpha- psychophysical testing, Western blots, indirect immu- cyano-4-hydroxycinnaminic acid (5 mg/mL in 50% acetoni- nohistologic investigations, and proteomic analysis to trile, 0.1% trifluoroacetic acid [TFA], and 2mM ammonium ci- identify the antigenic retinal proteins. Careful family his- trate) matrix was added to the digested peptides. The extracts tories were taken for autoimmune disorders to clarify the were evaporated to dryness and then dissolved in 5 µL of 60% genetic autoimmune backgrounds of patients with MAR. acetonitrile, 0.1% TFA. A 0.5-µL volume of this solution was spotted on a 192-well matrix-assisted laser desorption ioniza- tion target and allowed to dry. METHODS Mass spectra were acquired using tandem time-of-flight mass spectrometry(4800 Proteomics Analyzer; Applied Biosys- CLINICAL EXAMINATIONS tems, Foster City, California). Database searching was per- formed using commercially available software (GPS Explorer Eleven patients with histories of melanoma and subsequent reti- version 3.6, with Mascot version 2.1 against International Pro- nopathies of unknown origins and 8 patients with CAR and AIR tein Index human version 3.32 using the following para- (included for comparison) were examined at the retinal dys- meters: 50-ppm mass tolerance, 1 missed cleavage, carbami- trophy clinic at the Kellogg Eye Center, University of Michi- domethyl (C) fixed modification, and variable modifications gan, Ann Arbor. The patients were evaluated using standard- pyroglu (N-term Q) and oxidation (M). Spectra were acquired ized kinetic visual fields, electroretinographic testing, fundus in tandem mass spectrometry 2-kV–positive mode. photography, and clinical examinations. Serum samples were obtained after informed consent and were stored at −80°C un- IMMUNOBLOTS AND SDS–POLYACRYLAMIDE til studied. As control subjects, 9 healthy volunteers with nor- GEL ELECTROPHORESIS mal ERGs and no family history of autoimmune diseases were recruited. Informed consent was obtained from all patients and Retinal extract (20 µg of proteins), 0.1 µg of aldolase A, 0.1 µg control subjects. The experimental protocol was approved by of carbonic anhydrase II (CAII), 0.4 µg of aldolase C, 0.2 µg of the University of Michigan institutional review board. recoverin, 0.2 µg of S-arrestin, 0.4 µg of S-transferase (GST) fu- sion ␣-enolase, and 0.2 µg of heat shock protein 60 (HSP60)