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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Arch Pathol Lab Med. Author manuscript; available in PMC 2010 August 1.
Published in final edited form as:
PMCID: PMC2810483
NIHMSID: NIHMS166579

Anterior Diffuse Retinoblastoma Mutational Analysis and Immunofluorescence Staining

Abstract

Retinoblastoma is the most common primary intraocular tumor of childhood and may be heritable or occur sporadically. Anterior diffuse retinoblastoma is an uncommon variant that is thought to be sporadic. We describe a child with anterior diffuse retinoblastoma who presented with a pseudohypopyon. Genetic analysis showed a germline mutation of the RB1 allele that is potentially heritable. Immunofluorescence staining was positive for transforming growth factor β and for vascular endothelial growth factor and negative for inducible nitric oxide synthase and for hypoxia inducible factor α in the tumor seeds, indicating acquisition of nonischemia-mediated survival factors of the tumor seeds in the aqueous humor.

Retinoblastoma is the most common primary intraocular tumor in childhood. When a family history is present, 50% of offspring of a parent carrying an RB1 mutant allele will inherit that allele; 40% of them will develop retinoblastoma tumors. Each tumor that forms will have lost the second RB1 allele. Diffuse anterior retinoblastoma presents in older children and may mimic uveitis because it presents as cells in the posterior compartment and anterior chamber without an obvious retinal mass. Diffuse anterior retinoblastoma has previously been described as occurring in sporadic mutations. Herein, we report on a patient with diffuse anterior retinoblastoma who is the first member of a family to carry a germline RB1 mutant allele. Immunofluorescence staining showed that tumor seeds in the aqueous humor and implanted in the iris expressed nonischemia-related survival factors transforming growth factor β (TGF-β) and vascular endothelial growth factor (VEGF).

REPORT OF A CASE

A 9-year-old girl was evaluated for blurry vision in her left eye. The patient was otherwise healthy, and there was no family history of eye disease. The patient’s mother had noted redness and changes in color of the left eye during the preceding 2 months. Examination showed 20/20 vision in the right eye and 20/60 vision in the left eye. The intraocular pressures were 16 and 34 mm Hg in the right and left eyes, respectively. Examination showed a normal right eye. Anterior segment examination showed a pseudohypopyon in the left eye (Figure 1). Dilated fundus examination showed a possible small, inferior, peripheral mass in her left retina. An anterior-chamber fine-needle aspiration biopsy showed a small, round blue cell tumor, consistent with retinoblastoma (Figure 2, A and B). Complete blood cell count and bone marrow biopsy were performed, and findings from both were normal. The left eye was enucleated. Mutational analysis of the patient’s tumor showed 2 RB1 mutant alleles: a nonsense mutation c.763C → T(R255X) and a deletion, c.1572delA. The nonsense mutation was also identified in the DNA from the patient’s blood, indicating a germline mutation (Table).

Figure 1
The anterior chamber of the left eye contains a pseudohypopyon composed of white cells with associated neovascularization.
Figure 2
A, A fine-needle aspiration of the anterior chamber shows sheets of small, round, blue tumor cells. B, Some of these cells form rosettes (Wright-Giemsa, original magnification × 100).
Table thumbnail
RB1 Genetic Mutational Analysis of Tumor Sample and Patient’s Blood Sample

PATHOLOGIC FINDINGS

Gross examination of the enucleated eye showed white material in the anterior chamber. Similar white material was present anterior to the vitreous base and extended circumferentially for 360° (Figure 3, A). A small, white tumor was present in the peripheral retina near the ora serrata inferiorly. Scattered retinal hemorrhages with white centers were present (Figure 3, B and C). Microscopic examination showed fibrovascular tissue on the anterior surfaces of the iris leaflets causing ectropion uveae. The tumor was present in the anterior chamber and implanted into the iris (Figure 4, A). Islands of tumor and individual tumor cells were present in the space between the vitreous base and ciliary body (Figure 4, B). Tumor cells extended within the aqueous humor, through the pupil, around the iris, and into the anterior chamber. There were occasional Homer Wright rosettes in the tumor, especially in the islands of tumor in the anterior chamber. The peripheral inferior retina contained a 3 × 1 mm focus of tumor. The tumor was composed of small, round blue cells with high nuclear to cytoplasmic ratios and hyper-chromatic nuclei. There was a hemorrhage associated with the intraretinal tumor. The tumor was confined to the retina and overlying aqueous by the anterior hyaloid face of the vitreous by a tamponade effect of the vitreous humor (Figure 4, C). The tumor did not invade into the subretinal pigment epithelium space or into the choroid. Immunofluorescent staining for TGF-β1/2/3 (Santa Cruz Biotechnology, Santa Cruz, California), VEGF (Santa Cruz Biotechnology), inducible nitric oxide synthase (iNOS; R&D Systems, Minneapolis, Minnesota), and hypoxia inducible factor (HIF; Santa Cruz Biotechnology) was performed. Tumor seeds in the aqueous humor stained positively for TGF-β and VEGF (Figure 5, A through D). The retinal focus stained for VEGF alone. The tumor seeds and intra-retinal tumor failed to stain for iNOS or HIF1α.

Figure 3
Gross anatomic pathology of the enucleated eye. A, There is 360° tumor seeding of the space between the vitreous base and ciliary body. B and C, Areas of retinal hemorrhages with central white spots.
Figure 4
A, Tumor cells are present in the anterior chamber, trabecular meshwork, on the iris surface, and within the iris, where there is neovascularization (arrow). B, Tumor cells are present in the aqueous humor between the ciliary epithelium and the anterior ...
Figure 5
Immunostaining for tumor growth factor β (TGF-β; A and B) and immunostaining for vascular endothelial growth factor (VEGF; C and D). Counterstain for all (A through D) was propidium iodide. A, Immunofluorescent staining is positive in ...

COMMENT

Growth patterns of retinoblastoma can be endophytic, exophytic, or diffuse. Most retinoblastomas exhibit both exophytic and endophytic components. Anterior diffuse retinoblastoma, a variant of diffuse retinoblastoma, results in 360° seeding in the area of the vitreous base/ciliary body with an associated anterior chamber pseudohypopyon.13 The diffuse growth pattern results in late clinical diagnoses because there is no retinal mass, and associated tumor seeding may mimic uveitis. Children with diffuse infiltrating retinoblastoma are usually diagnosed between ages 5 and 12 years, with a mean age at diagnosis of 6.1 years.4 One case of diffuse retinoblastoma showed loss of heterozygosity in diffuse retinoblastoma cells.5 There is an inconspicuous, peripheral focus of intraretinal retinoblastoma in anterior diffuse retinoblastoma, which results in shedding of tumor cells into the aqueous humor between the ciliary epithelium and vitreous base, with the aqueous humor carrying the tumor into the anterior chamber. This form of retinoblastoma is unilateral and has been thought to be nonheritable. Our case exhibited a germline mutation that has been previously reported in heritable retinoblastoma,6 although it is unclear whether that case was an isolated unilateral tumor or a multifocal and bilateral tumor.

To better understand the cell signaling and survival mechanisms in this unusual variant of retinoblastoma, we performed immunofluorescent staining for TGF-β, VEGF, iNOS, and HIF. Tumor growth factor β exhibits tumor suppression via effects on proliferation, replication potential, and apoptosis. Tumor growth factor β also exhibits tumor promotion via effects on migration, invasion, angiogenesis, and the immune system. The dual nature of TGF-β is dependent on the cell type and genetic status of proteins in the signal transduction pathway.7 Interestingly, RB1 can regulate TGF-β gene expression via cyclin-dependent kinase inhibitors, depending on the cell type.8 Vascular endothelial growth factor is a member of the tyrosine kinase platelet–derived growth factor superfamily and includes placental growth factor, VEGF-A through VEGF-E. Vascular endothelial growth factor has been mapped to 6pter–p21 and is a multifunctional cytokine involved with angiogenesis, vasculogenesis, and cell survival.9 Inducible nitric oxide synthase generates nitric oxide from L-arginine, modulates malignant transformation, angiogenesis, metastasis; mediates angiogenesis via VEGF and basic fibroblast growth factor; and is expressed in ischemic retina, where it mediates intraretinal to intravitreal angiogenesis.10,11 Hypoxia inducible factor 1 is the primary mediator of low oxygen-tension environments (hypoxia) leading to angiogenesis.12 The tumor seeds in the aqueous humor in our case expressed TGF-β and VEGF and did not express iNOS or HIF1. The intraretinal tumor expressed VEGF, which did not appear to be mediated by HIF1 or iNOS, the most common pathways of ischemia mediated angiogenesis. We speculate that the tumor expression of VEGF was likely not due to ischemia, and tumor seeds acquired the expression of TGF-β, a survival factor, in the aqueous humor.

Anterior diffuse retinoblastoma is a variant of diffuse retinoblastoma that arises as a small focus in the peripheral retina. Free tumor cells express TGF-β and VEGF, are spread via the aqueous humor between the vitreous base and ciliary epithelium for 360°, migrate through the pupil where they become implanted into the iris, and cause neovascularization of the iris. This is a microcosm of neoplastic transformation, wherein tumors acquire the ability for limitless replication, evasion of apoptosis, and production of self-sufficient growth signals; become insensitive to antigrowth signals; produce sustained angiogenesis; and invade tissue via metastasis.13 In our case, there was a germline mutation, indicating that this form of retinoblastoma may be heritable.

Acknowledgments

This study was supported, in part, by grant EY06360 from the National Institutes of Health, and an unrestricted departmental grant from Research to Prevent Blindness, Inc. Several pictures and technical details were acquired with the significant aid of Weiqing Gao, BS. We are deeply grateful for her assistance in the preparation of this manuscript.

Footnotes

The authors have no relevant financial interest in the products or companies described in this article.

References

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