Meta-analysis in metastatic uveal melanoma to determine progression free and overall survival benchmarks: an international rare cancers initiative (IRCI) ocular melanoma study.

BACKGROUND: Despite the completion of numerous phase II studies, a standard of care treatment has yet to be defined for metastatic uveal melanoma (mUM). To determine benchmarks of progression free survival (PFS) and overall survival (OS), we carried out a meta-analysis using individual patient level trial data. METHODS: Individual patient variables and survival outcomes were requested from 29 trials published from 2000 to 2016. Univariable and multivariable analysis were carried out for prognostic factors. The variability between trial arms and between therapeutic agents on PFS and OS was investigated. RESULTS: OS data were available for 912 patients. The median PFS was 3.3 months (95% CI 2.9-3.6) and 6-month PFS rate was 27% (95% CI 24-30). Univariable analysis showed male sex, elevated (i.e. > versus ≤ upper limit of normal) lactate dehydrogenase (LDH), elevated alkaline phosphatase (ALP) and diameter of the largest liver metastasis (≥3 cm versus <3 cm) to be substantially associated with shorter PFS. Multivariable analysis showed male sex, elevated LDH and elevated ALP were substantially associated with shorter PFS. The most substantial factors associated with 6-month PFS rate, on both univariable and multivariable analysis were elevated LDH and ALP. The median OS was 10.2 months (95% CI 9.5-11.0) and 1 year OS was 43% (95% CI 40-47). The most substantial prognostic factors for shorter OS by univariable and multivariable analysis were elevated LDH and elevated ALP. Patients treated with liver directed treatments had statistically significant longer PFS and OS. CONCLUSION: Benchmarks of 6-month PFS and 1-year OS rates were determined accounting for prognostic factors. These may be used to facilitate future trial design and stratification in mUM.

Training in cataract surgery in Spain: analysis of the results of a survey of the European Board of Ophthalmology in a Spanish cohort.

INTRODUCTION: A survey conducted by the European Board of Ophthalmology (EBO) revealed significant differences in the surgical training of the ophthalmology residents in Europe, including a disparity between the sexes and a variation in the experience on cataract surgery (CC) between them. This study is about the Spanish sub-cohort of the survey, and its objective is to present and analyse the peculiarities of ophthalmology training in Spain within the European context, as well as discussing ways to harmonise and improve that training throughout the EU. METHODS: We analyse data of the Spanish participants in the EBO exams, defining subgroups by the Autonomous Communities existing in Spain. RESULTS: 93 of 135 requested participants (68.9%) responded. A 60.2% passed the EBO exam between 2021 and 2022, being mostly women (65.59%) aged 31 years old on average. The 91.4% were right-handed, coming from 13 of the 17 Spanish autonomous communities, although mostly from the Community of Valencia, Madrid and Catalonia. Respectively, 16.1%, 3.2% and 8.7% of the respondents said they have completed 10 or more training sessions on animal eyes, synthetic eyes and through the virtual reality simulator. This training was correlated with greater self-confidence in the management of a posterior capsular tear during surgery (p .025). All respondents manifested to have already performed stages of the CC. The average number of operations reported was 181.6 with regional disparities. A significant difference is observed between the sexes against women (-28.3%, p 0.03). DISCUSSION: Ophthalmologists in Spain, much more than other European countries, have greater opportunities for surgical training, with surgical procedures during the residency, that nearly triples those made by the others. Spanish women refer, like their European colleagues, to be in disadvantage in learning opportunities about cataract surgery. The Simulation Based Medical Education (SBME) allows to respond to the training deficit and complements the training on the patient. Although we demonstrate a significant correlation between the number of procedures carried out and self-confidence to operate simple cases, the SBME would be a complementary tool in self-confidence in front of a complication like capsular rupture. CONCLUSION: Spain massively adopts the model named by us "surgery for all", despite the underrepresentation of women in this area, emphasising a need for cultural change that the SBME could facilitate.

Mutations in KERA, encoding keratocan, cause cornea plana.

Specialized collagens and small leucine-rich proteoglycans (SLRPs) interact to produce the transparent corneal structure. In cornea plana, the forward convex curvature is flattened, leading to a decrease in refraction. A more severe, recessively inherited form (CNA2; MIM 217300) and a milder, dominantly inherited form (CNA1; MIM 121400) exist. CNA2 is a rare disorder with a worldwide distribution, but a high prevalence in the Finnish population. The gene mutated in CNA2 was assigned by linkage analysis to 12q (refs 4, 5), where there is a cluster of several SLRP genes. We cloned two additional SLRP genes highly expressed in cornea: KERA (encoding keratocan) in 12q and OGN (encoding osteoglycin) in 9q. Here we report mutations in KERA in 47 CNA2 patients: 46 Finnish patients are homozygous for a founder missense mutation, leading to the substitution of a highly conserved amino acid; and one American patient is homozygous for a mutation leading to a premature stop codon that truncates the KERA protein. Our data establish that mutations in KERA cause CNA2. CNA1 patients had no mutations in these proteoglycan genes.

Reduction of intra-ocular pressure by ocular compression before combined peri- and retrobulbar block.

The intra-ocular pressure immediately before glaucoma surgery can be raised. We wished to investigate if ocular compression for 20 min before a combined peri- and retrobulbar injection would result in a lower pressure after the block. Sixty consecutive patients scheduled for filtration surgery were randomly assigned to receive ocular compression using an external pressure device for 20 min before combined peri- and retrobulbar injection (intervention group, who also received compression after the block) or to a control group in whom pressure was applied only after the block was completed. The intra-ocular pressure was measured at baseline, after the 20-min pre-injection compression (intervention group), after injecting the block, and after the 10-min post-injection compression. The pressure did not differ between groups at baseline, after the block or after the post-injection compression. In the intervention group, the compression before the block reduced the median (IQR [range]) pressure from 21.0 (17.0-25.0 [12.0-40.0]) mmHg to 16.8 (12.5-22.5 [7.5-33.5]) mmHg (p<0.001). We conclude that external ocular compression reduces the intra-ocular pressure, but applying an additional compression for 20 min before injecting the block is not beneficial.

[The IC3D classification of the corneal dystrophies]. / IC3D-Klassifikation von Hornhautdystrophien.

BACKGROUND: The recent availability of genetic analyses has demonstrated the shortcomings of the current phenotypic method of corneal dystrophy classification. Abnormalities in different genes can cause a single phenotype, whereas different defects in a single gene can cause different phenotypes. Some disorders termed corneal dystrophies do not appear to have a genetic basis. PURPOSE: The purpose of this study was to develop a new classification system for corneal dystrophies, integrating up-to-date information on phenotypic description, pathologic examination, and genetic analysis. METHODS: The International Committee for Classification of Corneal Dystrophies (IC3D) was created to devise a current and accurate nomenclature. RESULTS: This anatomic classification continues to organize dystrophies according to the level chiefly affected. Each dystrophy has a template summarizing genetic, clinical, and pathologic information. A category number from 1 through 4 is assigned, reflecting the level of evidence supporting the existence of a given dystrophy. The most defined dystrophies belong to category 1 (a well-defined corneal dystrophy in which a gene has been mapped and identified and specific mutations are known) and the least defined belong to category 4 (a suspected dystrophy where the clinical and genetic evidence is not yet convincing). The nomenclature may be updated over time as new information regarding the dystrophies becomes available. CONCLUSIONS: The IC3D Classification of Corneal Dystrophies is a new classification system that incorporates many aspects of the traditional definitions of corneal dystrophies with new genetic, clinical, and pathologic information. Standardized templates provide key information that includes a level of evidence for there being a corneal dystrophy. The system is user-friendly and upgradeable and can be retrieved on the website www.corneasociety.org/ic3d .

Primary lymphoma of the lacrimal sac: an EORTC ophthalmic oncology task force study.

AIM: To define the clinical and histopathological characteristics of primary lacrimal sac lymphoma in a predominantly white population. METHODS: Specimens of lacrimal sac lymphoma and follow up data were solicited from members of the Ophthalmic Oncology Task Force of the European Organization for Research and Treatment of Cancer (EORTC) and the European Ophthalmic Pathology Society (EOPS). Specimens were stained with haematoxylin and eosin and an immunohistochemical panel against leucocyte antigens was applied. Diagnosis was reached by consensus of five experienced pathologists according to the World Health Organization classification system. The histopathological findings were correlated with the clinical data. RESULTS: Of 15 primary lacrimal sac lymphomas, five (33%) were diffuse large B cell lymphoma (DLBCL), five (33%) were extranodal marginal zone B cell lymphoma of mucosa associated lymphoid tissue (MALT lymphoma), three were classified as "transitional MALT lymphoma," being in transition from MALT lymphoma to DLBCL, and two were unclassified B cell lymphomas. Nine of the patients were female, and the median age at the time of diagnosis was 71 years (range 45-95 years). The most frequent presenting symptoms were epiphora (85%), swelling in the region of the lacrimal sac (79%), and dacryocystitis (21%). All but one patient presented in stage I. Systemic spread occurred in three of nine patients (33%). The 5 year overall survival was 65%. CONCLUSIONS: DLBCL and MALT lymphoma are equally common in the lacrimal sac in contrast with the remaining periorbital and/or orbital region where MALT lymphoma predominates.

Microvascular loops and networks as prognostic indicators in choroidal and ciliary body melanomas.

BACKGROUND: Malignant melanoma of the ciliary body and choroid of the eye is a tumor that disseminates frequently, and 50% of the diagnosed patients die within 10 years. We investigated the hypothesis that, by histopathologic analysis of the arrangement of microvessels (i.e., small blood vessels) in loops and networks, we might be able to differentiate better those patients with a favorable prognosis from those with a poor prognosis. METHODS: We conducted a population-based, retrospective cohort study of melanoma-specific and all-cause mortality for 167 consecutive patients who had an eye surgically removed because of malignant choroidal or ciliary body melanoma during the period from 1972 through 1981. Microvascular loops and networks were evaluated independently by two pathologists who were unaware of patient outcome. RESULTS: Microvascular patterns could be assessed in 134 (80%) of 167 melanoma specimens. The 10-year probability of melanoma-specific survival was worse if microvascular loops (0.45 versus 0.83; two-sided P<.0001) and networks (0.41 versus 0.72, two-sided P<.0001) were present. In multivariate Cox regression analysis of melanoma-specific survival, the hazard ratios were 1.66 (95% confidence interval [CI] = 1.19-2.30) for the presence of loops and networks as a combined three-category variable, 2.36 (95% CI = 1.37-4.05) for the presence of epithelioid cells, 1.11 (95% CI = 1.03-1.19) for the largest basal tumor diameter (evaluated as a continuous variable), and 2.14 (95% CI = 1.25-3.67) for ciliary body involvement. CONCLUSIONS: Patients with malignant uveal melanoma who have a favorable prognosis can be distinguished from those with a poor prognosis by histopathologic analysis of microvascular patterns in uveal melanoma tumor specimens.

Trilateral retinoblastoma: a meta-analysis of hereditary retinoblastoma associated with primary ectopic intracranial retinoblastoma.

PURPOSE: To obtain refined knowledge regarding trilateral retinoblastoma (TRb), which is a syndrome that consists of hereditary retinoblastoma associated with an intracranial neuroblastic tumor. MATERIALS AND METHODS: Using a systematic literature review, we contacted authors to obtain missing information. Data from 106 children were used in a meta-analysis including frequency distributions and Kaplan-Meier survival curves. RESULTS: TRb showed no sex predilection. Median age at diagnosis of retinoblastoma was 5 months (range, 0 to 29 months); age at diagnosis was younger among 47 children (47%) with familial retinoblastoma compared with age at diagnosis among 52 children (53%) with sporadic retinoblastoma (2 v 6.5 months, P <.0001). TRb usually affected the second or third generation with retinoblastoma. Median time from retinoblastoma to TRb was 21 months (range, 6 months before to 141 months after); time to TRb was longer for 78 (77%) pineal tumors compared with 23 (23%) suprasellar tumors (32 v 6.5 months, P <.0001). The size (27 v 32 mm, P =.57) and prognosis (survival of 9 v 8 months, P =.91) of pineal and suprasellar tumors were similar. TRb was detected earlier (1 v 22 months, P =.0007) and the child survived longer if neuroimaging was routinely performed (16 v 8 months, P =.001), but age at death was similar (36 v 37 months, P =.98). Cumulative 5-year survival (which was likely to indicate cure) was 27% (v 0%) if screening was undertaken. All children whose TRb exceeded 15 mm in size died. CONCLUSION: The family history, age at diagnosis, and laterality of retinoblastoma in children with TRb resembled that of ordinary hereditary retinoblastoma. Suprasellar TRb were diagnosed earlier, and may arise earlier, than pineal TRb. Screening by neuroimaging could improve the cure rate if cases of TRb were detected when tumors were 15 mm or smaller in size.

The HNK-1 carbohydrate epitope in the eye: basic science and functional implications.

The HNK-1 carbohydrate epitope is part of many cell membrane and extracellular matrix molecules. It has been implicated in cell to cell and cell to extracellular matrix adhesion, and antibodies to the HNK-1 epitope are emerging as a versatile tool in eye research. They have been used to identify a novel cell type in the human eye, the subepithelial matrix cells that reside in the inner connective tissue layer (ICTL) of the ciliary body. Although these cells resemble fibroblasts in ultrastructure, they form a distinct cell population that differs in its antigenic profile from fibroblasts of other tissues. These cells are associated with the elastic fiber system of the ICTL. Other structures in the human eye that harbor the HNK-1 epitope in a nonrandom pattern are the ciliary and iris epithelia, the zonular lamella, the lens capsule, the retina, glial cells of the optic and ciliary nerves, and scleral fibroblasts. The HNK-1 epitope in the eye appears early during embryonic development and is phylogenetically conserved, but many interspecies differences exist in its distribution. The role of the HNK-1 epitope may be to structurally stabilize the ciliary body and the retina, and to participate in zonular attachments. The HNK-1 epitope has been linked with many common eye diseases. The subepithelial matrix cells seem to be susceptible to undergo irreversible damage as a result of glaucoma, thermal injury, and tissue compression. This epitope has proved to be useful in identifying intraocular deposits of exfoliation syndrome. It can explain the adhesiveness of exfoliation material. Intraocular exfoliation material differs in HNK-1 immunoreactivity from the extraocular fibrillopathy of exfoliation syndrome and its presence in fellow eyes also argues against the concept of unilateral exfoliation syndrome. The HNK-1 epitope is found in the extracellular matrix of secondary cataract and anterior subcapsular cataract, and it may contribute to their pathogenesis. Finally, the HNK-1 epitope can be used to trace neuroepithelial derivatives of the optic vesicle in developmental anomalies and in tumors of the eye. Eventual identification of molecules that bear the HNK-1 epitope in the eye will likely shed light on many aspects of ocular physiology and pathobiology

Exfoliation syndrome and exfoliation glaucoma.

Exfoliation syndrome abnormal deposition in the anterior segment of the eye of an unknown substance thought to be related to elastic fibres and basement membrane components is associated with accelerated cataract progression. increased frequency of intraoperative and postoperative complications and increased risk for glaucoma and. therefore, is a clinically important finding. A clear association has been shown with age. The syndrome occurs worldwide but its prevalence seems to vary from country to country. The best-known sign of exfoliation syndrome is deposits of greyish-white material on the anterior lens surface. Sometimes exfoliation material can also be seen at the pupillary border, on the anterior iris surface, corneal endothelium, and on the anterior vitreous face. When clinically detected, exfoliation syndrome is somewhat more often unilateral than bilateral. According to recent investigations clinically unilateral exfoliation syndrome is probably never truly unilateral but rather asymmetric, because exfoliation material has been detected ultrastructurally and immunohistochemically around iris blood vessels of the nonexfoliative fellow eyes. Indeed, electron microscopy identifies in various organs of patients with exfoliation syndrome fibrils similar to those seen in intraocular exfoliation deposits. Other clinical signs associated with exfoliation syndrome are pigment dispersion, transillumination defects of the iris and reduced response to mydriatics. In unilateral exfoliation syndrome, intraocular pressure (IOP) of the exfoliative eye is approximately 2 mmHg higher than IOP of the nonexfoliative fellow eye. Whether elevated IOP, vascular changes or exfoliation syndrome itself is the main factor causing optic nerve head damage and conversion of an exfoliative eye to glaucomatous, is not known. Glaucoma in the exfoliation syndrome has been shown to have a more serious clinical course than in primary open-angle glaucoma (POAG). At the time of diagnosis, IOP and its diurnal variation are generally higher and visual field defects tend to be greater in exfoliation glaucoma than in POAG. Because the decrease in lOP variation and lowering of the mean IOP level has been shown to improve visual field prognosis more in exfoliation glaucoma than in POAG, the glaucomatous process is considered to be more pressure-related in exfoliation glaucoma. Furthermore, progression of optic disc damage has been shown to be similar in exfoliation glaucoma and POAG when lOPs are lowered to a comparable level by the treatment. However, vascular disturbances in the posterior segment of the eye might after all be of equal importance in these two types of glaucoma; optic disc haemorrhages and venous occlusions have been reported to be as frequent in exfoliation glaucoma as in POAG. Perhaps in exfoliation glaucoma circullatory disturbances combined with high IOP lead to a particularly relentlessly progressing form of the disease.

Structure, development and function of cytoskeletal elements in non-neuronal cells of the human eye.

The cytoskeleton, of which the main components in the human eye are actin microfilaments, intermediate filaments and microtubules with their associated proteins, is essential for the normal growth, maturation, differentiation, integrity and function of its cells. These components interact with intra- and extracellular environment and each other, and their profile frequently changes during development, according to physiologic demands, and in various diseases. The ocular cytoskeleton is unique in many ways. A special pair of cytokeratins, CK 3 and 12, has apparently evolved only for the purposes of the corneal epithelium. However, other cytokeratins such as CK 4, 5, 14, and 19 are also important for the normal ocular surface epithelia, and other types may be acquired in keratinizing diseases. The intraocular tissues, which have a relatively simple cytoskeleton consisting mainly of vimentin and simple epithelial CK 8 and 18, differ in many details from extraocular ones. The iris and lens epithelium characteristically lack cytokeratins in adults, and the intraocular muscles all have a cytoskeletal profile of their own. The dilator of the iris contains vimentin, desmin and cytokeratins, being an example of triple intermediate filament expression, but the ciliary muscle lacks cytokeratin and the sphincter of the iris is devoid even of vimentin. Conversion from extraocular-type cytoskeletal profile occurs during fetal life. It seems that posttranslational modification of cytokeratins in the eye may also differ from that of extraocular tissues. So far, it has not been possible to reconcile the cytoskeletal profile of intraocular tissues with their specific functional demands, but many theories have been put forward. Systematic search for cytoskeletal elements has also revealed novel cell populations in the human eye. These include transitional cells of the cornea that may represent stem cells on migration, myofibroblasts of the scleral spur and juxtacanalicular tissue that may modulate aqueous outflow, and subepithelial matrix cells of the ciliary body and myofibroblasts of the choroid that may both participate in accommodation. In contrast to the structure and development of the ocular cytoskeleton, changes that take place in ocular disease have not been analysed systematically. Nevertheless, potentially meaningful changes have already been observed in corneal dystrophies (Meesmann's dystrophy, posterior polymorphous dystrophy and iridocorneal endothelial syndrome), degenerations (pterygium) and inflammatory diseases (Pseudomonas keratitis), in opacification of the lens (anterior subcapsular and secondary cataract), in diseases characterized by proliferation of the retinal pigment epithelium (macular degeneration and proliferative vitreoretinopathy), and in intraocular tumours (uveal melanoma). In particular, upregulation of alpha-smooth muscle actin seems to be a relatively general response typical of spreading and migrating corneal stromal and lens epithelial cells, trabecular cells and retinal pigment epithelial cells.

Expression of intermediate filaments (IF) in tissues and cultured cells.

Intermediate filaments are found in most nucleated cells as part of their cytoskeleton. Intermediate filaments are formed by different proteins in cells of major tissues types. Therefore, antibodies against intermediate filaments can be used in tissue typing, in the analysis of cell lineages during development and in the elucidation of the origin of unknown tumors.

Bleomycin, vincristine, lomustine and dacarbazine (BOLD) in combination with recombinant interferon alpha-2b for metastatic uveal melanoma.

This EORTC multicentre study analysed the efficacy and tolerability in patients with metastatic uveal melanoma of BOLD chemotherapy in combination with recombinant interferon alpha-2b. The dose of bleomycin was 15 mg on days 2 and 5, of vincristine 1 mg/m(2) on days 1 and 4, of lomustine 80 mg on day 1, and of dacarbazine (DTIC) 200 mg/m(2) on days 1-5, given every 4 weeks for a minimum of two cycles. Subcutaneous (s.c.) interferon alpha-2b at a dose of 3 x 10(6) IU was initiated on day 8 of the first cycle, and continued at a dose of 6 x 10(6) IU three times per week after 6 weeks. A median of two cycles were administered to 24 patients (median age 60.5 years). None achieved an objective response (0%; 95% Confidence Interval (CI): 0-14), 2 (8.3%) remained stable, 20 showed progression, and 2 (8.3%) were invaluable. The median progression-free survival was 1.9 months (95% CI: 1.8-3.4) and overall survival 10.6 months (95% CI: 6.9-16.4). Overall survival improved with increasingly favourable pretreatment characteristics (median, 14.7 versus 6.9 versus 6.0 months for Helsinki University Central Hospital (HUCH) Working Formulation stages IVBa, IVBb and IVBc, respectively; P=0.018). Grade 3 alopecia and neurotoxicity occurred in 13% of the patients. This multicentre study did not confirm earlier reports that BOLD with human leucocyte or recombinant interferon would induce at least 15% objective responses in metastatic uveal melanoma.

Antigenic profile of the human lacrimal gland.

The antigenic profile of 13 normal formalin-fixed, paraffin-embedded human main and accessory lacrimal glands, biopsied from patients aged 11 to 78 years, was studied using a panel of 27 polyclonal and monoclonal antibodies. Secretory cells of lacrimal acini reacted with antibodies to S-100 protein and simple epithelium-type cytokeratins CK 7, CK 8, CK 18, and CK 19. Their luminal membranes were labeled with antibodies to carcinoembryonic antigen, epithelial membrane antigen, and epithelial glycoproteins recognized by Ber-EP4. Myoepithelial cells were often immunopositive for S-100 protein, vimentin, glial fibrillary acidic protein (GFAP), and alpha-smooth muscle actin. More rarely, they reacted with antibodies recognizing CK 5, CK 13, and CK 14, which consistently labeled the basal cells of lacrimal ducts. Unlike myoepithelial cells, basal ductal cells were immunopositive for CK 7, CK 8, CK 18, and CK 19. In main excretory ducts, dendritic melanocyte-like cells co-expressing vimentin and S-100 protein intermingled with ductal epithelial cells. The luminal cells of lacrimal ducts basically paralleled secretory cells in their antigenic profile, although they lacked Ber-EP4 and were immunopositive for CK 4. Antibodies to neuron-specific enolase and synaptophysin reacted with nerve fibers among negatively reacting secretory acini. This antigenic profile closely parallels that of salivary glands and provides a basis for studies of lacrimal gland pathology.

Cytoskeleton in neuroectodermally derived epithelial and muscle cells of the human iris and ciliary body.

The cytoskeleton of epithelial and muscle cells of the human iris and ciliary body was analyzed by immunohistochemistry in three morphologically normal formalin-fixed, paraffin-embedded eyes and in 34 eyes containing a uveal melanoma. Both layers of the iris epithelium reacted with monoclonal antibodies (MAb) V9 and Vim 3B4 to vimentin, whereas the ciliary epithelia additionally reacted with MAb CAM 5.2, CK5, KS-B17.2, and CY-90, recognizing cytokeratins 8 and 18. The same cytokeratin MAb labeled the retinal pigment epithelium, which lacked vimentin. The muscle portion of the anterior iris epithelium, which forms the dilator muscle, as well as the sphincter and ciliary muscles, reacted with MAb DE-U-10 to desmin and 1A4 to alpha-smooth muscle actin. The dilator and ciliary muscles also reacted with V9 and Vim 3B4 to vimentin, and some dilator fibers were weakly immunopositive for cytokeratin 8 and 18 with CY-90 and CAM 5.2. The antigenic profile of iris and ciliary epithelia infiltrated by melanoma cells remained unchanged. The intraocular epithelia, which are developmentally related but differ in function, and the intraocular muscles, which differ in origin but are functionally related, have distinct cytoskeletal profiles and may provide insights into the functional significance of intermediate filament expression.

Parvalbumin, a horizontal cell-associated calcium-binding protein in retinoblastoma eyes.

PURPOSE: To search for differentiation in retinoblastoma toward horizontal cells and retinal neurons other than photoreceptor cells with antibodies to parvalbumin, a horizontal, ganglion, and amacrine cell-associated antigen. METHODS: Fifty formalin-fixed and paraffin-embedded human eyes with an intraocular retinoblastoma and two orbital recurrences were studied using the avidin-biotinylated peroxidase complex method and monoclonal antibody (mAb) PA-235 to parvalbumin. RESULTS: In the retinas of retinoblastoma eyes obtained after birth, horizontal cells at the outer border of the inner nuclear layer and their processes in the inner part of the outer plexiform layer always reacted with mAb PA-235. Immunolabeled ganglion and amacrine cells were found, respectively, in 31 (76%; 95% confidence interval [CI] 60-88) and 15 (37%; 95% (CI 22-53) of the 41 eyes with preserved retinas, whereas bipolar and photoreceptor cells were unlabeled. Undifferentiated and differentiated retinoblastoma cells in all studied specimens were negative for parvalbumin. However, immunopositive horizontal and ganglion cells engulfed by the tumor were present within 36 of the 50 retinoblastomas (72%; 95% CI 58 - 84), which often allowed the tracing of otherwise invisible remnants of former infiltrated retinas. CONCLUSIONS: Parvalbumin is a useful marker for horizontal and ganglion cells in normal and pathologic human retinas, including those entrapped within retinoblastoma. The absence of parvalbumin from tumor cells argues against differentiation similar to that seen in these parvalbumin-positive neurons and subpopulations of amacrine cells from the second trimester onward.

Development of cytoskeleton in neuroectodermally derived epithelial and muscle cells of the human eye.

PURPOSE: To analyze the cytoskeletal development in neuroectodermally derived epithelial and muscle cells of the human eye during the second and third trimesters of pregnancy. METHODS: Nine formalin-fixed, paraffin-embedded fetal autopsy eyes from the 13th to 40th week of gestation were studied with eight monoclonal antibodies (mAbs) to intermediate filaments and alpha-smooth muscle actin (alpha SMA) by the avidin-biotinylated-peroxidase complex method. RESULTS: The epithelium of the iris reacted with mAbs Vim 3B4 and V9 to vimentin in all eyes. Reactivity with mAb CAM 5.2 to cytokeratin (CK) 8 and mAb CY-90 and KS-B17.2 to CK 18 disappeared from the iris epithelium by the 28th gestational week. mAb 1A4 to alpha SMA labeled its anterior layer from the 28th week on. mAbs DE-U-10 and D33 to desmin labeled dilator fibers by the 37th week, and focal reactivity for CK 8 and 18 concurrently appeared. The developing iris sphincter reacted for alpha SMA in all eyes. It was labeled increasingly for desmin from the 18th week on, whereas initial reactivity for vimentin gradually disappeared after the 22nd week. mAbs to vimentin, CK 8, and CK 18 labeled the ciliary epithelium and the retinal pigment epithelium in all eyes, except for lack of CK 8 and 18 in the nonpigmented ciliary epithelium at the 13th week. The ciliary muscle reacted uniformly for vimentin and alpha SMA and from the 16th week on for desmin. CONCLUSIONS: The results highlight the individuality of cytoskeletal profiles in the neuroectodermal epithelial and muscle cells of the eye and clarify the formation of the peculiar cytoskeleton typical of the adult human eye. They also offer a framework for detecting cytoskeletal changes in developmental anomalies of the eye after the first trimester of pregnancy.

Intermediate filaments in the human retina and retinoblastoma. An immunohistochemical study of vimentin, glial fibrillary acidic protein, and neurofilaments.

Fifty-five retinoblastoma specimens were studied by a sensitive immunoperoxidase method to determine the intermediate filament types present in human retina and retinoblastoma. Polyclonal antiserum against vimentin and monoclonal antibodies to glial fibrillary acidic protein (GFAP) and to the 200 kD neurofilament triplet protein were used. In the human retina, Müller's cells coexpressed vimentin and GFAP in most instances, probably as a reactive phenomenon. Surprisingly, the horizontal cells did not stain with any of the antibodies used, and may thus lack intermediate filaments. Also, the meshwork of neural fibers in the inner plexiform layer was unusually sparse. Retinoblastoma cells were found to be devoid of all intermediate filament types studied. The tumors contained, however, vimentin and GFAP in the stromal cells. All neurofilament-positive cells in retinoblastoma apparently derived from infiltrated retina. One retinoblastoma eye was also studied by indirect immunofluorescence microscopy of frozen sections with identical results.

Synaptophysin in the human retina and retinoblastoma. An immunohistochemical and Western blotting study.

Fifty-four formalin-fixed and paraffin-embedded intraocular retinoblastoma specimens and three human eyes enucleated because of orbital tumors were studied for the presence of synaptophysin, a neuron-associated integral membrane glycoprotein of presynaptic vesicles, by using the monoclonal antibody SY38. Normal human brain was used as control. In the human retina, synaptophysin-like immunoreactivity was present in both plexiform layers, but could not be detected in neuronal perikarya. However, in reactive retinas present in retinoblastoma eyes, synaptophysin was often observed in perikarya and processes of photoreceptors. Positive neoplastic cells were found in 45 of the 54 retinoblastomas. Differentiated tumors tended to contain greater numbers of positive cells than undifferentiated ones, a third of which were entirely negative. Identical immunoreactivity was seen in frozen specimens from human retina and from three retinoblastomas. Using Western blotting, a major polypeptide comigrating with human brain synaptophysin was detected in human retina, and a similar but slightly slower migrating band in retinoblastoma. The results support a primarily neuronal origin for this tumor and point to the possibility that synaptic elements, previously observed in a few cases, may be more frequent in retinoblastoma than had been thought.

Identification of a novel element in the human eye: the inner connective tissue layer of the ciliary body characterized with antibodies to the HNK-1 epitope.

PURPOSE: To characterize the nature and the developmental distribution of the HNK-1 epitope in the inner connective tissue layer of the human ciliary body, located between the ciliary epithelium and muscle with two monoclonal antibodies to the HNK-1 epitope common to many cell adhesion molecules. METHODS: Nine fetal (gestational age 13-40 wk) and 32 postnatal human eyes (age 3 mo to 78 yr) were studied by immunohistochemistry with monoclonal antibodies HNK-1 and VC1.1 to the HNK-1 epitope. Antibodies to cytoskeletal elements were used to characterize the cells in this region. RESULTS: The HNK-1-immunopositive cells appeared underneath the pigment epithelium of the pars plicata by the 20th gestational week, spread into the pars plana after the 28th week, and reached the ora serrata during the first year of life. The immunoreaction was constantly present in all adult eyes examined; they were sharply demarcated from the iris, ciliary muscle, and choroid. The HNK-1-positive subepithelial layer was not labeled with monoclonal antibodies V9 or Vim 3B4 to vimentin, monoclonal antibodies CAM 5.2 and CY-90 to cytokeratin 8 and 18, or monoclonal antibodies DE-U-10 and D33 to desmin in adult eyes, but was uniformly positive for vimentin in fetal eyes. The HNK-1 epitope was distributed along cell membranes or adjacent extracellular matrix of stromal cells. CONCLUSION: The HNK-1-positive stromal region is a constant and conspicuous element of the human eye that may have a role in structurally stabilizing the ciliary body, perhaps in relation to accommodation or aqueous secretion.