David S. Bardenstein

Collaborative Ocular Oncology Group report number 1: prospective validation of a multi-gene prognostic assay in uveal melanoma.

PURPOSE This study evaluates the prognostic performance of a 15 gene expression profiling (GEP) assay that assigns primary posterior uveal melanomas to prognostic subgroups: class 1 (low metastatic risk) and class 2 (high metastatic risk). DESIGN Prospective, multicenter study. PARTICIPANTS A total of 459 patients with posterior uveal melanoma were enrolled from 12 independent centers. TESTING Tumors were classified by GEP as class 1 or class 2. The first 260 samples were also analyzed for chromosome 3 status using a single nucleotide polymorphism assay. Net reclassification improvement analysis was performed to compare the prognostic accuracy of GEP with the 7th edition clinical Tumor-Node-Metastasis (TNM) classification and chromosome 3 status. MAIN OUTCOME MEASURES Patients were managed for their primary tumor and monitored for metastasis. RESULTS The GEP assay successfully classified 446 of 459 cases (97.2%). The GEP was class 1 in 276 cases (61.9%) and class 2 in 170 cases (38.1%). Median follow-up was 17.4 months (mean, 18.0 months). Metastasis was detected in 3 class 1 cases (1.1%) and 44 class 2 cases (25.9%) (log-rank test, P<10(-14)). Although there was an association between GEP class 2 and monosomy 3 (Fisher exact test, P<0.0001), 54 of 260 tumors (20.8%) were discordant for GEP and chromosome 3 status, among which GEP demonstrated superior prognostic accuracy (log-rank test, P = 0.0001). By using multivariate Cox modeling, GEP class had a stronger independent association with metastasis than any other prognostic factor (P<0.0001). Chromosome 3 status did not contribute additional prognostic information that was independent of GEP (P = 0.2). At 3 years follow-up, the net reclassification improvement of GEP over TNM classification was 0.43 (P = 0.001) and 0.38 (P = 0.004) over chromosome 3 status. CONCLUSIONS The GEP assay had a high technical success rate and was the most accurate prognostic marker among all of the factors analyzed. The GEP provided a highly significant improvement in prognostic accuracy over clinical TNM classification and chromosome 3 status. Chromosome 3 status did not provide prognostic information that was independent of GEP.

EPHA2 Is Associated with Age-Related Cortical Cataract in Mice and Humans

Age-related cataract is a major cause of blindness worldwide, and cortical cataract is the second most prevalent type of age-related cataract. Although a significant fraction of age-related cataract is heritable, the genetic basis remains to be elucidated. We report that homozygous deletion of Epha2 in two independent strains of mice developed progressive cortical cataract. Retroillumination revealed development of cortical vacuoles at one month of age; visible cataract appeared around three months, which progressed to mature cataract by six months. EPHA2 protein expression in the lens is spatially and temporally regulated. It is low in anterior epithelial cells, upregulated as the cells enter differentiation at the equator, strongly expressed in the cortical fiber cells, but absent in the nuclei. Deletion of Epha2 caused a significant increase in the expression of HSP25 (murine homologue of human HSP27) before the onset of cataract. The overexpressed HSP25 was in an underphosphorylated form, indicating excessive cellular stress and protein misfolding. The orthologous human EPHA2 gene on chromosome 1p36 was tested in three independent worldwide Caucasian populations for allelic association with cortical cataract. Common variants in EPHA2 were found that showed significant association with cortical cataract, and rs6678616 was the most significant in meta-analyses. In addition, we sequenced exons of EPHA2 in linked families and identified a new missense mutation, Arg721Gln, in the protein kinase domain that significantly alters EPHA2 functions in cellular and biochemical assays. Thus, converging evidence from humans and mice suggests that EPHA2 is important in maintaining lens clarity with age.

Retinoblastoma. Immunohistochemistry and cell differentiation.

Tumor from eight enucleated eyes was analyzed by immunohistochemistry, using a panel of specific antibodies including interphotoreceptor retinoid-binding protein (IRBP), S-antigen (S-Ag), opsin, neuron-specific enolase (NSE), glial fibrillary acidic protein (GFAP), laminin, and vimentin. In addition, immunoelectron microscopy and enzyme-linked immunosorbent assay (ELISA) for IRBP were performed. Immunohistochemical staining disclosed the most pronounced labeling of tumor cells with NSE and IRBP antibodies. A correlation was found between the degree of tumor differentiation and amount of IRBP, a protein specifically synthesized by photoreceptor cells. Moderate labeling of the better differentiated tumors was also observed with antibodies against S-Ag and focal labeling in a few tumors with opsin antibodies. Anti-GFAP labeling was limited to a smaller number of reactive glial cells and perivascular glial cells. These data indicate the essential neuronal nature of retinoblastoma tumor cells in situ as well as at least partial photoreceptor-like features, as shown by the presence of recognized photoreceptor cell markers (IRBP, S-Ag, opsin). Tissue culture studies using the human Y-79 retinoblastoma cell line also demonstrate that the tumor cells are primitive multipotential retinoblasts capable of at least partial differentiation along neuronal, glial, or pigment epithelial cell lines.

A multicenter study to map genes for Fuchs endothelial corneal dystrophy: Baseline characteristics and heritability

Purpose: To describe the methods for family and case–control recruitment for a multicenter genetic and associated heritability analyses of Fuchs endothelial corneal dystrophy (FECD). Methods: Twenty-nine enrolling sites with 62 trained investigators and coordinators gathered individual and family information, graded the phenotype, and collected blood and/or saliva for genetic analysis on all individuals with and without FECD. The degree of FECD was assessed in a 0 to 6 semiquantitative scale using standardized clinical methods with pathological verification of FECD on at least 1 member of each family. Central corneal thickness was measured by ultrasonic pachymetry. Results: Three hundred twenty-two families with 330 affected sibling pairs with FECD were enrolled and included a total of 650 sibling pairs of all disease grades. Using the entire 7-step FECD grading scale or a dichotomous definition of severe disease, heritability was assessed in families via sib–sib correlations. Both binary indicators of severe disease and semiquantitative measures of disease severity were significantly heritable, with heritability estimates of 30% for severe disease, 37% to 39% for FECD score, and 47% for central corneal thickness. Conclusions: Genetic risk factors have a strong role in the severity of the FECD phenotype and corneal thickness. Genotyping this cohort with high-density genetic markers followed by appropriate statistical analyses should lead to novel loci for disease susceptibility.

Differing Roles for TCF4 and COL8A2 in Central Corneal Thickness and Fuchs Endothelial Corneal Dystrophy

Fuchs endothelial corneal dystrophy (FECD) is the most common late-onset, vision-threatening corneal dystrophy in the United States, affecting about 4% of the population. Advanced FECD involves a thickening of the cornea from stromal edema and changes in Descemet membrane. To understand the relationship between FECD and central corneal thickness (CCT), we characterized common genetic variation in COL8A2 and TCF4, genes previously implicated in CCT and/or FECD. Other genes previously associated with FECD (PITX2, ZEB1, SLC4A11), and genes only known to affect CCT (COL5A1, FOXO1, AVGR8, ZNF469) were also interrogated. FECD probands, relatives and controls were recruited from 32 clinical sites; a total of 532 cases and 204 controls were genotyped and tested for association of FECD case/control status, a 7-step FECD severity scale and CCT, adjusting for age and sex. Association of FECD grade with TCF4 was highly significant (OR  = 6.01 at rs613872; p = 4.8×10−25), and remained significant when adjusted for changes in CCT (OR  = 4.84; p = 2.2×10−16). Association of CCT with TCF4 was also significant (p = 6.1×10−7), but was abolished with adjustment for FECD grade (p = 0.92). After adjusting for FECD grade, markers in other genes examined were modestly associated (p ∼ 0.001) with FECD and/or CCT. Thus, common variants in TCF4 appear to influence FECD directly, and CCT secondarily via FECD. Additionally, changes in corneal thickness due to the effect of other loci may modify disease severity, age-at-onset, or other biomechanical characteristics.

Localization of the complement membrane attack complex inhibitor (CD59) in human conjunctiva and lacrimal gland

Recent studies have established that complement is present in the eye and participates in ocular defense. The mechanisms by which ocular tissues are protected from bystander injury arising from local activation of the cascade, however, have not been characterized. Decay accelerating factor (DAF or CD55) and the membrane inhibitor of reactive lysis (MIRL or CD59) are cell surface regulatory proteins that protect blood cells from uptake of autologous C3b and polymerization of autologous C9 on their surfaces. In previous studies, we found that DAF is expressed in high levels on corneal, conjunctival, and lacrimal gland acinar surfaces. In this study we assayed ocular and lacrimal gland tissues for CD59. Immunohistochemical analyses demonstrated large amounts of the protein the same locations. The presence of CD59 in these sites is consistent with the proposal that CD59 functions together with DAF in protecting ocular tissues from autologous complement-mediated injury.

Upregulation of DAF (CD55) on orbital fibroblasts by cytokines. Differential effects of TNF-ß and TNF-a

Purpose. Decay accelerating factor (DAF) and membrane cofactor protein (MCP) are membrane complement regulators that protect self cells from deposition of autologous C3b on their surfaces. CD59, a third downstream regulator of the cascade, prevents the assembly on self cells of autologous membrane-attack complexes. All three proteins are highly expressed on corneal and conjunctival epithelia, and are present in lower levels on multiple intraocular and adnexal cell types. The purpose of this study was to determine whether, and if so, how DAF, MCP and CD59 expression by ocular and adnexyl cells is modulated by cytokines. Methods. Primary cultures of orbital fibroblasts and corneal epithelial cells were incubated with TNF-a, TNF-ß, TGF-ß1, IFN-?, MIF or blocking anti-MIF mABs and extracts of the cells quantitated for DAF, MCP and CD59 by two-site immunoradiometric assays. Where inductions occurred, the kinetics of the increases, the effect of combining cytokines, and the effect of protein kinase-C inhibition were studied. Results. DAF expression on orbital fibroblasts was upregulated 6.3-, 3.7- and 4.2-fold by TGF-ß1, TNF-ß and IFN-?, respectively, but that its expression on corneal epithelial cells was minimally affected. These same (or other) cytokines did not significantly upregulate MCP or CD59. The cytokine-induced upregulation of DAF expression on orbital fibroblasts requires 24 hr for IFN-? or 48 hr for TGF-ß1 or TNF-ß, is dependent on new protein synthesis, and does not involve protein kinase-C activation. Conclusions. TGF-ß1-, TNF-ß- and IFN-?-mediated upregulation of DAF should serve to prevent complement-mediated injury to orbital fibroblasts in the course of ocular inflammation. The induction by TNF-ß rather than TNF-a contrasts with that on all other cell types studied.

Ki-67 and bromodeoxyuridine labeling of human choroidal melanoma cells

Understanding tumor growth patterns has implications for prognosis as well as for response and susceptibility to treatment. The antibody Ki-67 was used as a marker of cycling cells and bromodeoxyuridine (BrdUrd) was used as a marker of proliferating cells to characterize the cycling and proliferative rates of cells from human choroidal melanoma. The BrdUrd labeling indices varied from 0-1.1% and the Ki-67 labeling indices ranged from 0-3.0.3%. Linear regression modeling showed good correlation defined by the equation: Ki-67 index = 0.237 + 1.63 x BrdUrd labeling index with r = 0.919. Correlations between these indices and clinical and histologic parameters were not significant.

Tears contain the complement regulator CD59 as well as decay-accelerating factor (DAF)

Previous studies have shown that DAF (or CD55), a cell surface inhibitor of autologous C3 activation, is present in tears and that > 90% of the C3 convertase regulatory activity in tear fluid resides in this protein (Lass JH et al., Invest Ophth Vis Sci 1990; 31:1136–48). This study investigated whether (i) the membrane cofactor protein (MCP or CD46), an additional factor that regulates C3 activation, and (ii) the membrane inhibitor of reactive lysis (MIRL or CD59), a cell surface regulator that acts to prevent formation of the membrane attack complex, are also present in tears, and if so, are functional. Two‐site immunoradiometric assays showed that MCP is present in tears at low levels (42 + 8 ng/ml, n = 8) while CD59 is present at levels (222 + 78 ng/ml, n = 14) comparable to those of DAF (325 + 289 ng/ml, n = 12). The concentrations of CD59 (i) were increased two‐fold or more in closed eye tears, and (ii) were decreased in reflex tears. Western blotting showed that CD59 protein in tears migrates with an apparent mol. wt similar to membrane CD59 protein. Phenyl–Sepharose adsorption and Triton X‐114 partitioning of tear CD59 as well as of tear DAF however, showed that both proteins are devoid of GPI anchors. Assays using cobra venom factor‐activated human serum and guinea pig erythrocytes showed that CD59 is functionally active in inhibiting autologous C5b‐9‐mediated lysis and, under constitutive conditions, accounts for > 85% of the C9 inhibitory activity in tear fluid.

Hyperfocal Cryotherapy of Multiple Molluscum contagiosum Lesions in Patients with the Acquired Immune Deficiency Syndrome

BACKGROUND Patients with the acquired immune deficiency syndrome are at risk for the development of multiple lesions of Molluscum contagiosum on the eyelids. In this setting, traditional methods of treatment frequently are ineffective and pose risks to the patient as well as to the treating physician. METHODS A technique was developed that combined lidocaine/prilocaine topical anesthesia with hyperfocal cryotherapy. Twelve patients with multiple M. contagiosum lesions of the eyelids were treated. Initially, two methods were used: one application for 30 seconds or two applications for 20 seconds. RESULTS Lesions treated with two 20-second applications regressed. Most of those lesions treated for 30 seconds regressed. No scarring, lash complications, ptosis, or damage to the underlying cornea or deeper ocular structures was observed in any patient. CONCLUSIONS Hyperfocal cryotherapy is an effective therapy for multiple M. contagiosum lesions of the periorbital region, posing minimal risk to the patient and physician. It is particularly useful in patients who are positive for the human immunodeficiency virus, who frequently have multiple lesions, are likely to have recurrent disease, and who pose risks of disease transmission to the medical personnel caring for them.