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Anterior diffuse retinoblastoma: mutational analysis and immunofluorescence staining.

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 p (TGRp) and vascular endothelial growth factor (VEGF).


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 [right arrow] 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).


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[degrees] (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 X 1 mm focus of tumor. The tumor was composed of small, round blue cells with high nuclear to cytoplasmic ratios and hyperchromatic 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-[beta] 1/2/3 (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-[beta] and VEGF (Figure 5, A through D). The retinal focus stained for VEGF alone. The tumor seeds and intraretinal tumor failed to stain for iNOS or HIF1a.



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[degrees] seeding in the area of the vitreous base/ciliary body with an associated anterior chamber pseudohypopyon. (1-3) 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) Thereisan 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-[beta], VEGF, iNOS, and HIF. Tumor growth factor p exhibits tumor suppression via effects on proliferation, replication potential, and apoptosis. Tumor growth factor [beta] also exhibits tumor promotion via effects on migration, invasion, angiogenesis, and the immune system. The dual nature of TGF-[beta] is dependent on the cell type and genetic status of proteins in the signal transduction pathway. (7) Interestingly, RB1 can regulate TGF-[beta] 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-[beta] 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-[beta], 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-[beta] and VEGF, are spread via the aqueous humor between the vitreous base and ciliary epithelium for 360[degrees], migrate through the pupil where they become implanted into the iris, and cause neo vascularization 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.


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.


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Michelle B. Crosby, MD, PhD; G. Baker Hubbard, MD; Brenda L. Gallie, MD; Hans E. Grossniklaus, MD, MBA

Accepted for publication March 31, 2009.

From the Department of Ophthalmology, Emory University School of Medicine, Atlanta, Georgia (Drs Crosby, Hubbard, and Grossniklaus); and the Division of Applied Molecular Oncology, Ontario Cancer Institute, Princess Margaret Hospital, Ontario, California (Dr Gallie).

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

Correspondence: Hans E. Grossniklaus, MD, L. F. Montgomery Laboratory, BT 428, Emory Eye Center, 1 365 Clifton Rd, Atlanta, GA 30322 (e-mail:
RB1 Genetic Mutational Analysis of Tumor Sample and Patient's Blood

                       Allele 1                 Allele 2

Tumor                  c.763.C   T(R255)X       c1572delA
Blood                  c.763.C   T(R255)X       Normal findings

Mutation description   Exon 8 causing an        Heterozygous deletion
                       immediate termination    of the A nucleotide in
                       codon                    exon 17

Effect on protein      Termination codon        A frameshift leading
                       leading to a truncated   to a termination codon
                       nonfunctional Rb         and truncated,
                       protein                  nonfunctional Rb

Abbreviation: Rb, retinoblastoma.
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Article Details
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Author:Crosby, Michelle B.; Hubbard, G. Baker; Gallie, Brenda L.; Grossniklaus, Hans E.
Publication:Archives of Pathology & Laboratory Medicine
Article Type:Disease/Disorder overview
Geographic Code:1USA
Date:Aug 1, 2009
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